Anti-fc epsilon-r1 alpha (fcer1a) antibodies, bispecific antigen-binding molecules that bind fcer1a and cd3, and uses thereof

ABSTRACT

The present invention provides novel full-length human antibodies that bind to human Fc epsilon-R1 alpha (monospecific antibodies). The present invention also provides novel bispecific antibodies (bsAbs) that bind to both Fc epsilon-R1 alpha and CD3 and activate T cells via the CD3 complex in the presence of Fc epsilon-R1 alpha-expressing cells. The bispecific antigen-binding molecules of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced Fc epsilon-R1 alpha-targeted immune response is desired and/or therapeutically beneficial. For example, the bispecific antibodies of the invention are useful for the treatment of allergies, including anaphylaxis.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/547,910, filed on Aug. 22, 2019, which is related to andclaims priority to U.S. Provisional Application No. 62/721,921, filed onAug. 23, 2018. The entire contents of each of the foregoing applicationsare expressly incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jan. 10, 2023, isnamed 10480_118003-45304_SL and is 94,341 bytes in size. The sequencelisting is part of the specification and is incorporated in its entiretyby reference herein.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for FcεR1α, and methods of usethereof. The present invention also relates to bispecificantigen-binding molecules that bind FcεR1α and CD3, and methods of usethereof.

BACKGROUND

FcεR1 is a high affinity Fc receptor for Immunoglobulin E (IgE), andFcεR1 binds to IgE with an equilibrium dissociation constant (K_(D))value of about 10⁻¹⁰ M. FcεR1 receptor crosslinking by allergen-boundIgE leads to cellular degranulation and subsequent allergic response,and the serum level of IgE is positively correlated with FcεR1.

Human FcεR1 is expressed in mast cells, basophils, monocytes,macrophages, mDCs, pDCs, Langerhans cells, eosinophils and platelets.Mast cells and basophils are innate effector cells that play a role inallergy and anaphylaxis via allergen mediated crosslinking of the IgEreceptor, FcεR1α. Other roles include wound healing and mucosalimmunity.

There are two types of human multimeric cell surface FcεR1 receptors,the tetrameric form and the trimeric form. The tetrameric human FcεR1comprises an α chain, a β chain, and a homodimer of γ chains (αβγ₂), andthe trimeric human FcεR1 comprises an α chain and a homodimer of γchains (αγ₂). The α-chain of FcεR1 binds to a single IgE antibodymolecule, while there is no reported role for β- and γ-chains in ligandbinding.

Human FcεR1 binds to both human and murine IgE, and interleukin-4 (IL-4)enhances the expression of the α-chain in humans. In contrast, murineFcεR1 only has the tetrameric αβγ₂ isoform and is expressed in mastcells and basophils. IL-4 does not enhance the expression of the α-chainof murine FcεR1.

CD3 is a homodimeric or heterodimeric antigen expressed on T cells inassociation with the T cell receptor complex (TCR) and is required for Tcell activation. Functional CD3 is formed from the dimeric associationof two of four different chains: epsilon, zeta, delta and gamma. The CD3dimeric arrangements include gamma/epsilon, delta/epsilon, andzeta/zeta. Antibodies against CD3 have been shown to cluster CD3 on Tcells, thereby causing T cell activation in a manner similar to theengagement of the TCR by peptide-loaded MHC molecules. Thus, anti-CD3antibodies have been proposed for therapeutic purposes involving theactivation of T cells.

Antigen-binding molecules that target FcεR1α, as well as bispecificantigen-binding molecules that bind both FcεR1α and CD3 would be usefulin therapeutic settings in which specific targeting and T cell-mediatedkilling of cells that express FcεR1α is desired.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention provides antibodies andantigen-binding fragments thereof that bind to human FcεR1α. Theantibodies according to this aspect are useful, inter alia, fortargeting cells expressing FcεR1α. The present invention also providesbispecific antibodies and antigen-binding fragments thereof that bindhuman FcεR1α and human CD3. The bispecific antibodies according to thisaspect are useful, inter alia, for targeting T cells expressing CD3, andfor stimulating T cell activation, e.g., under circumstances where Tcell-mediated killing of cells expressing FcεR1α is beneficial ordesirable. For example, the bispecific antibodies can directCD3-mediated T cell activation to specific FcεR1α-expressing cells, suchas mast cells or basophils.

Exemplary anti-FcεR1α antibodies of the present invention are listed inTables 1 and 2 herein. Table 1 sets forth the amino acid sequenceidentifiers of the heavy chain variable regions (HCVRs) and light chainvariable regions (LCVRs), as well as heavy chain complementaritydetermining regions (HCDR1, HCDR2 and HCDR3), light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3), heavychain (HC), and light chain (LC) of the exemplary anti-FcεR1αantibodies. Table 2 sets forth the sequence identifiers of the nucleicacid molecules encoding the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1,LCDR2, LCDR3, HC and LC of the exemplary anti-FcεR1α antibodies.

The present invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR comprising an amino acid sequence selectedfrom any of the HCVR amino acid sequences listed in Table 1, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an LCVR comprising an amino acid sequenceselected from any of the LCVR amino acid sequences listed in Table 1, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCVR and an LCVR amino acid sequencepair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listedin Table 1 paired with any of the LCVR amino acid sequences listed inTable 1. According to certain embodiments, the present inventionprovides antibodies, or antigen-binding fragments thereof, comprising anHCVR/LCVR amino acid sequence pair contained within any of the exemplaryanti-FcεR1α antibodies listed in Table 1. In certain embodiments, theHCVR/LCVR amino acid sequence pair is of SEQ ID NOs: 2/26, 10/26, or18/26 (e.g., mAb17110, mAb17111, or mAb17112)

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising anamino acid sequence selected from any of the HCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising anamino acid sequence selected from any of the HCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising anamino acid sequence selected from any of the HCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR1 (LCDR1) comprising anamino acid sequence selected from any of the LCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR2 (LCDR2) comprising anamino acid sequence selected from any of the LCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR3 (LCDR3) comprising anamino acid sequence selected from any of the LCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequencepair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequenceslisted in Table 1 paired with any of the LCDR3 amino acid sequenceslisted in Table 1. According to certain embodiments, the presentinvention provides antibodies, or antigen-binding fragments thereof,comprising an HCDR3/LCDR3 amino acid sequence pair contained within anyof the exemplary anti-FcεR1α antibodies listed in Table 1. In certainembodiments, the HCDR3/LCDR3 amino acid sequence pair is of SEQ ID NOs:8/32, 16/32, or 24/32 (e.g., mAb17110, mAb17111, or mAb17112).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a set of six CDRs (i.e.,HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of theexemplary anti-FcεR1α antibodies listed in Table 1. In certainembodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acidsequences set is selected from the group consisting of SEQ ID NOs:4-6-8-28-30-32, 12-14-16-28-30-32, or 20-22-24-28-30-32 (e.g., mAb17110,mAb17111, or mAb17112).

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a set of six CDRs (i.e.,HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR aminoacid sequence pair as defined by any of the exemplary anti-FcεR1αantibodies listed in Table 1. For example, the present inventionincludes antibodies, or antigen-binding fragments thereof, comprisingthe HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences setcontained within an HCVR/LCVR amino acid sequence pair of SEQ ID NOs:2/26, 10/26, or 18/26 (e.g., mAb17110, mAb17111, or mAb17112). Methodsand techniques for identifying CDRs within HCVR and LCVR amino acidsequences are well known in the art and can be used to identify CDRswithin the specified HCVR and/or LCVR amino acid sequences disclosedherein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

The present invention also provides nucleic acid molecules encodinganti-FcεR1α antibodies or portions thereof. For example, the presentinvention provides nucleic acid molecules encoding any of the HCVR aminoacid sequences listed in Table 1; in certain embodiments the nucleicacid molecule comprises a polynucleotide sequence selected from any ofthe HCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-FcεR1α antibodies listedin Table 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-FcεR1α antibodies listedin Table 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-FcεR1α antibody listed in Table1.

The present invention also provides recombinant expression vectorscapable of expressing a polypeptide comprising a heavy or light chainvariable region of an anti-FcεR1α antibody. For example, the presentinvention includes recombinant expression vectors comprising any of thenucleic acid molecules mentioned above, i.e., nucleic acid moleculesencoding any of the HCVR, LCVR, and/or CDR sequences as set forth inTable 1. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antibodies or portions thereof by culturing thehost cells under conditions permitting production of the antibodies orantibody fragments, and recovering the antibodies and antibody fragmentsso produced.

The present invention includes anti-FcεR1α antibodies having a modifiedglycosylation pattern. In some embodiments, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afructose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds FcεR1α and a pharmaceutically acceptable carrier. Ina related aspect, the invention features a composition which is acombination of an anti-FcεR1α antibody and a second therapeutic agent.In one embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-FcεR1α antibody. Additionalcombination therapies and co-formulations involving the anti-FcεR1αantibodies of the present invention are disclosed elsewhere herein.

In another aspect, the invention provides therapeutic methods fortargeting/killing FcεR1α-expressing cells (e.g., mast cells, orbasophils) using an anti-FcεR1α antibody of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising an anti-FcεR1αantibody of the invention to a subject in need thereof. In some cases,the anti-FcεR1α antibodies (or antigen-binding fragments thereof) can beused for treating allergy, or may be modified to be more cytotoxic bymethods, including but not limited to, modified Fc domains to increaseADCC (see e.g. Shield et al. (2002) JBC 277:26733), radioimmunotherapy,antibody-drug conjugates, or other methods for increasing the efficiencyof FcεR1α expressing cells killing.

The present invention also includes the use of an anti-FcεR1α antibodyof the invention in the manufacture of a medicament for the treatment ofa disease or disorder (e.g., allergy) related to or caused byFcεR1α-expressing cells.

In yet another aspect, the invention provides monospecific anti-FcεR1αantibodies for diagnostic applications, such as, e.g., imaging reagents.

In yet another aspect, the invention provides therapeutic methods forstimulating T cell activation using an anti-CD3 antibody orantigen-binding portion of an antibody of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising an antibody of thepresent invention.

In another aspect, the present invention provides an isolated antibodyor antigen-binding fragment thereof that binds human FcεR1α or bindscynomolgus (Macaca fascicularis) FcεR1α with a binding dissociationequilibrium constant (K_(D)) of less than about 250 nM as measured in asurface plasmon resonance assay at 25° C. In yet another aspect, thepresent invention provides an isolated antibody or antigen-bindingfragment thereof that binds human FcεR1α with a dissociative half-life(t½) of greater than about 0.54 minute or binds cynomolgus FcεR1α with adissociative half-life (t½) of greater than about 0.6 minute as measuredin a surface plasmon resonance assay at 25° C.

The invention further provides an antibody or antigen-binding fragmentthat competes for binding to human FcεR1α with a reference antibodycomprising an HCVR/LCVR amino acid sequence pair as set forth inTable 1. In another aspect, the invention provides an antibody orantigen-binding fragment that competes for binding to human FcεR1α witha reference antibody comprising an HCVR/LCVR amino acid sequence pairselected from the group consisting of SEQ ID NOs: 2/26; 10/26; and18/26.

The invention furthermore provides an antibody or antigen-bindingfragment, wherein the antibody or antigen-binding fragment thereof bindsto the same epitope on human FcεR1α as a reference antibody comprisingan HCVR/LCVR amino acid sequence pair as set forth in Table 1. Inanother aspect, the antibody or antigen-binding fragment binds to thesame epitope on human FcεR1α as a reference antibody comprising anHCVR/LCVR amino acid sequence pair selected from the group consisting ofSEQ ID NOs: 2/26; 10/26, and 18/26.

The invention further provides an isolated antibody or antigen-bindingfragment thereof that binds human FcεR1α, wherein the antibody orantigen-binding fragment comprises: the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR) having an aminoacid sequence as set forth in Table 1; and the CDRs of a light chainvariable region (LCVR) having an amino acid sequence as set forth inTable 1. In another aspect, the isolated antibody or antigen-bindingfragment comprises the heavy and light chain CDRs of an HCVR/LCVR aminoacid sequence pair selected from the group consisting of: 2/26; 10/26;and 18/26. In yet another aspect, the isolated antibody orantigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3domains, respectively, selected from the group consisting of: SEQ IDNOs: 4-6-8-28-30-32; 12-14-16-28-30-32; and 20-22-24-28-30-32

In another aspect, the invention provides an isolated antibody orantigen-binding fragment thereof that binds human FcεR1α, wherein theantibody or antigen-binding fragment comprises: (a) a heavy chainvariable region (HCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 10, and 18; and (b) a light chainvariable region (LCVR) having an amino acid sequence SEQ ID NO: 26. In afurther aspect, the isolated antibody or antigen-binding fragment anHCVR/LCVR amino acid sequence pair selected from the group consistingof: SEQ ID NOs: 2/26; 10/26; and 18/26.

According to another aspect, the present invention provides bispecificantigen-binding molecules (e.g., antibodies) that bind FcεR1α and CD3.Such bispecific antigen-binding molecules are also referred to herein as“anti-FcεR1α/anti-CD3 bispecific molecules,” “anti-CD3/anti-FcεR1αbispecific molecules,” “anti-FcεR1α×CD3,” “anti-CD3×FcεR1α,” or“FcεR1α×CD3 bsAbs.” The anti-FcεR1α portion of the anti-FcεR1α/anti-CD3bispecific molecule is useful for targeting cells that express FcεR1α(e.g., mast cells or basophils), and the anti-CD3 portion of thebispecific molecule is useful for activating T-cells. The simultaneousbinding of FcεR1α on a mast cells or basophils and CD3 on a T-cellfacilitates directed killing (cell lysis) of the targeted mast cells orbasophils by the activated T-cell. The anti-FcεR1α/anti-CD3 bispecificmolecules of the invention are therefore useful, inter alia, fortreating diseases and disorders related to or caused byFcεR1α-expressing cells (e.g., allergy).

The bispecific antigen-binding molecules according to this aspect of thepresent invention comprise a first antigen-binding domain thatspecifically binds human CD3, and a second antigen-binding domain thatspecifically binds FcεR1α. The present invention includesanti-FcεR1α/anti-CD3 bispecific molecules (e.g., bispecific antibodies)wherein each antigen-binding domain comprises a heavy chain variableregion (HCVR) paired with a light chain variable region (LCVR). Incertain exemplary embodiments of the invention, the anti-CD3antigen-binding domain and the anti-FcεR1α antigen binding domain eachcomprise different, distinct HCVRs paired with a common LCVR. Forexample, as illustrated in Example 1 herein, bispecific antibodies wereconstructed comprising a first antigen-binding domain that specificallybinds CD3, wherein the first antigen-binding domain comprises an HCVRand an LCVR, each derived from an anti-CD3 antibody; and a secondantigen-binding domain that specifically binds FcεR1α, wherein thesecond antigen-binding domain comprises an HCVR derived from ananti-FcεR1α antibody paired with the same LCVR. In such embodiments, thefirst and second antigen-binding domains comprise distinct anti-CD3 andanti-FcεR1α HCVRs but share a common LCVR. The amino acid sequence ofthis LCVR is shown, e.g., in SEQ ID NO: 26, and the amino acid sequencesof the corresponding CDRs (i.e., LCDR1-LCDR2-LCDR3) are shown in SEQ IDNOs: 28, 30, and 32, respectively. Genetically modified mice can be usedto produce fully human bispecific antigen-binding molecules comprisingtwo different heavy chains that associate with an identical light chainthat comprises a variable domain derived from one of two different humanlight chain variable region gene segments. Alternatively, variable heavychains may be paired with one common light chain and expressedrecombinantly in host cells. As such, the antibodies of the inventioncan comprise immunoglobulin heavy chains associated with a singlerearranged light chain. In some embodiments, the light chain comprises avariable domain derived from a human V_(K)1-39 gene segment or aV_(K)3-20 gene segment. In other embodiments, the light chain comprisesa variable domain derived from a human V_(K)1-39 gene segment rearrangedwith a human J_(K)5 or a human J_(K)1 gene segment (WO 2017/053856,herein incorporated by reference).

The present invention provides anti-CD3/anti-FcεR1α bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises any of the HCVR amino acid sequences, any of theLCVR amino acid sequences, any of the HCVR/LCVR amino acid sequencepairs, any of the heavy chain CDR1-CDR2-CDR3 amino acid sequences, orany of the light chain CDR1-CDR2-CDR3 amino acid sequences as set forthin US publication 2014/0088295 published Mar. 27, 2014 and WO2018/067331 published Apr. 12, 2018.

In addition, the present invention provides anti-CD3/anti-FcεR1αbispecific molecules, wherein the first antigen-binding domain thatspecifically binds CD3 comprises any of the HCVR amino acid sequences asset forth in Table 3 herein. The first antigen-binding domain thatspecifically binds CD3 may also comprise any of the LCVR amino acidsequences as set forth in Tables 1, and 3 herein. The present inventionalso provides anti-CD3/anti-FcεR1α bispecific molecules, wherein thefirst antigen-binding domain that specifically binds CD3 comprises anyof the heavy chain CDR1-CDR2-CDR3 amino acid sequences as set forth inTable 3, and/or any of the light chain CDR1-CDR2-CDR3 amino acidsequences as set forth in Tables 1, and 3 herein.

According to certain embodiments, the present invention providesanti-CD3/anti-FcεR1α bispecific molecules, wherein the firstantigen-binding domain that specifically binds CD3 comprises a heavychain variable region (HCVR) having an amino acid sequence as set forthin Table 1 herein or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-FcεR1α bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises a light chain variable region (LCVR) having an aminoacid sequence as set forth in Tables 1, and 3 herein, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-FcεR1α bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises an HCVR and LCVR (HCVR/LCVR) amino acid sequencepair as set forth in Table 3 herein.

The present invention also provides anti-CD3/anti-FcεR1α bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises a heavy chain CDR3 (HCDR3) domain having an aminoacid sequence as set forth in Table 3 herein, or a substantially similarsequence thereto having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a light chain CDR3 (LCDR3) domainhaving an amino acid sequence as set forth in Tables 1, and 3 herein, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the first antigen-binding domain thatspecifically binds CD3 comprises an HCDR3/LCDR3 amino acid sequence pairas set forth in Table 3 herein.

The present invention also provides anti-CD3/anti-FcεR1α bispecificantigen-binding molecules, wherein the first antigen-binding domain thatspecifically binds CD3 comprises a heavy chain CDR1 (HCDR1) domainhaving an amino acid as set forth in Table 3 herein, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domainhaving an amino acid as set forth in Table 3, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a heavy chain CDR3 (HCDR3) domain having anamino acid as set forth in Table 3, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; a light chain CDR1 (LCDR1) domain having an aminoacid sequence as set forth in Tables 1, and 3 herein, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; a light chain CDR2 (LCDR2) domainhaving an amino acid sequence as set forth in Tables 1, and 3 herein, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity, and a light chainCDR3 (LCDR3) domain having an amino acid sequence as set forth in Tables1, and 3 herein, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

Certain non-limiting, exemplary anti-CD3/anti-FcεR1α bispecificantigen-binding molecules of the invention include a firstantigen-binding domain that specifically binds CD3 comprisingHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences as set forth in Table 3 herein.

The present invention further provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain that specificallybinds human CD3 comprises heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region(HCVR) comprising an amino acid sequence as set forth in Table 3 andlight chain complementarity determining regions (LCDR1, LCDR2 and LCDR3)from a light chain variable region (LCVR) comprising an amino acidsequence as set forth in Tables 1, and 3.

In another aspect, the invention provides a bispecific antigen-bindingmolecule wherein the first antigen-binding domain that specificallybinds human CD3 comprises heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region(HCVR) comprising an amino acid sequence of SEQ ID NO: 42., and lightchain complementarity determining regions (LCDR1, LCDR2 and LCDR3) froma light chain variable region (LCVR) comprising an amino acid sequenceof SEQ ID NO: 26.

The invention further provides a bispecific antigen-binding molecule,wherein the first antigen-binding domain that specifically binds humanCD3 comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprising the aminoacid sequences of SEQ ID Nos: 44-46-48-28-30-32.

In a further aspect, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain that specificallybinds human CD3 comprises the heavy and light chain CDRs of an HCVR/LCVRamino acid sequence pair of SEQ ID NO: 42/26.

In more embodiments, exemplary anti-CD3/anti-FcεR1α bispecificantigen-binding molecules of the invention include a bispecificantigen-binding molecule wherein the first antigen-binding domain thatspecifically binds human CD3 comprises an HCVR comprisingHCDR1-HCDR2-HCDR3 having the amino acid sequences of SEQ ID NOs:44-46-48.

The present invention also provides anti-CD3/anti-FcεR1α bispecificmolecules, wherein the second antigen-binding domain that specificallybinds FcεR1α comprises a heavy chain variable region (HCVR) having theamino acid sequence selected from the group consisting of SEQ ID NOs: 2,10, and 18, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-FcεR1α bispecificmolecules, wherein the second antigen-binding domain that specificallybinds FcεR1α comprises a light chain variable region (LCVR) having theamino acid sequence of SEQ ID NO: 26, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides anti-CD3/anti-FcεR1α bispecificmolecules, wherein the second antigen-binding domain that specificallybinds FcεR1α comprises an HCVR and LCVR (HCVR/LCVR) amino acid sequencepair selected from the group consisting of SEQ ID NOs: 2/26, 10/26, and18/26.

The present invention also provides anti-CD3/anti-FcεR1α bispecificmolecules, wherein the second antigen-binding domain that specificallybinds FcεR1α comprises a heavy chain CDR3 (HCDR3) domain having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 8, 16,and 24, or a substantially similar sequence thereto having at least 90%,at least 95%, at least 98% or at least 99% sequence identity; and alight chain CDR3 (LCDR3) domain having an amino acid sequence of SEQ IDNO: 32, or a substantially similar sequence thereof having at least 90%,at least 95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the second antigen-binding domain thatspecifically binds FcεR1α comprises an HCDR3/LCDR3 amino acid sequencepair selected from the group consisting of SEQ ID NOs: 8/32, 16/32, and24/32.

The present invention also provides anti-CD3/anti-FcεR1α bispecificantigen-binding molecules, wherein the second antigen-binding domainthat specifically binds FcεR1α comprises a heavy chain CDR1 (HCDR1)domain having an amino acid sequence selected from the group consistingof SEQ ID NOs: 4, 12, and 20, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; a heavy chain CDR2 (HCDR2) domain having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 6, 14,and 22, or a substantially similar sequence thereof having at least 90%,at least 95%, at least 98% or at least 99% sequence identity; a heavychain CDR3 (HCDR3) domain having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 8, 16, and 24, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; a light chain CDR1 (LCDR1) domainhaving an amino acid sequence of SEQ ID NOs: 28, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domainhaving an amino acid sequence of SEQ ID NO: 30, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domainhaving an amino acid sequence of SEQ ID NOs: 32, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

Certain non-limiting, exemplary anti-CD3/anti-FcεR1α bispecificantigen-binding molecules of the invention include a secondantigen-binding domain that specifically binds FcεR1α comprisingHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences selected from the group consisting of: SEQ ID NOs:4-6-8-28-30-32, 12-14-16-28-30-32, and 20-22-24-28-30-32.

In a related embodiment, the invention includes anti-CD3/anti-FcεR1αbispecific antigen-binding molecules wherein the second antigen-bindingdomain that specifically binds FcεR1α comprises the heavy and lightchain CDR domains contained within heavy and light chain variable region(HCVR/LCVR) sequences selected from the group consisting of SEQ ID NOs:2/26, 10/26, and 18/26.

In another aspect, the invention provides a bispecific antigen-bindingmolecule comprising: (a) a first antigen-binding domain that comprisesthree heavy chain complementarity determining regions HCDR1, HCDR2 andHCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs:44, 46 and 48, and three light chain complementarity determining regionsLCDR1, LCDR2 and LCDR3, respectively, comprising the amino acidsequences of SEQ ID NOs: 28, 30 and 32, wherein the firstantigen-binding domain specifically binds human CD3; and (b) a secondantigen-binding domain that comprises three heavy chain complementaritydetermining regions (HCDR1, HCDR2 and HCDR3) and three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3); whereinHCDR1 comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NOs: 4, 12, and 20; HCDR2 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 6, 14, and22; HCDR3 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8, 16, and 24; LCDR1 comprises an amino acidsequence of SEQ ID NO: 28; LCDR2 comprises an amino acid sequence of SEQID NO: 30; and LCDR3 comprises an amino acid sequence of SEQ ID NO: 32,wherein the second antigen-binding arm specifically binds human FcεR1α.

In another aspect, the invention provides a bispecific antigen-bindingmolecule comprising a first antigen-binding domain that binds human CD3and a second antigen-binding domain that binds human FcεR1α, wherein thesecond antigen-binding domain is derived from the antibody orantigen-binding fragment of any one of the anti-FcεR1α antibodies of theinvention. In a further aspect, the invention provides a bispecificantigen-binding molecule comprising a first antigen-binding domain thatspecifically binds human CD3, and a second antigen-binding domain thatspecifically binds human FcεR1α.

The invention further provides a bispecific antigen-binding moleculewhich binds human cells expressing human CD3. In another aspect, thebispecific antigen-binding molecule binds human cells expressing humanFcεR1α and/or cells expressing cynomolgus FcεR1α.

In another aspect the invention provides a bispecific antigen-bindingmolecule which inhibits allergic reaction in a subject (e.g., mice)expressing human FcεR1α. The invention further provides bispecificantigen-binding molecules which deplete basophils or otherFcεR1α-expressing cells in a subject (e.g., mice) expressing humanFcεR1α.

In another aspect the invention provides a bispecific antigen-bindingmolecule comprising a second antigen-binding domain that specificallybinds a target cell expressing human FcεR1α with a binding ratio greaterthan 200 in the presence or absence of IgE or binds a target cellexpressing cynomolgus FcεR1α with a binding ratio greater than 140 inthe presence of absence of IgE, wherein such binding ratio is measuredin an in vitro FACS binding assay.

In some embodiments, the antigen-binding molecule induces Tcell-mediated killing of FcεR1α-expressing with an EC₅₀ value of lessthan about 20 nM, as measured in an in vitro T cell-mediated cellkilling assay, for example, where the FcεR1α expressing cells arebasophils.

In some applications, the second antigen-binding domain binds human orcynomolgus FcεR1α with a K_(D) value of less than about 467 nM, asmeasured in an in vitro surface plasmon resonance binding assay at 25°C. In some instances, the second antigen-binding domain binds each ofhuman FcεR1α and cynomolgus FcεR1α with an K_(D) value of less thanabout 450 nM, less than about 400 nM, less than about 350 nM, less thanabout 300 nM, less than about 250 nM, less than about 200 nM, less about150 nM, less than about 100 nM, or less than about 50 nM.

In certain embodiments, anti-FcεR1α antibodies of the invention,antigen-binding fragments and bispecific antibodies thereof were made byreplacing amino acid residues of a parental in a stepwise manner basedon differences between the germline sequence and the parental antibodysequence.

In another aspect, the present invention provides an isolated bispecificantigen-binding molecule that competes for binding to FcεR1α, or bindsto the same epitope on FcεR1α as a reference antibody, wherein thereference antibody comprises a first antigen-binding domain comprisingan HCVR/LCVR pair comprising the amino acid sequences of SEQ ID NOs:42/26, and a second antigen-binding domain comprising an HCVR/LCVR paircomprising the amino acid sequences of SEQ ID NOs: 2/26, 10/26 or 18/26.

In another aspect, the present invention provides an isolated bispecificantigen-binding molecule that competes for binding to human CD3, orbinds to the same epitope on human CD3 as a reference antibody, whereinthe reference antibody comprises a first antigen-binding domaincomprising an HCVR/LCVR pair comprising the amino acid sequences of SEQID NOs: 42/26, and a second antigen-binding domain comprising anHCVR/LCVR pair comprising the amino acid sequences of SEQ ID NOs: 2/26,10/26 or 18/26.

Any of the bispecific antigen-binding molecules discussed above orherein may be a bispecific antibody. In some embodiments, the bispecificantibody comprises a human IgG heavy chain constant region. In oneembodiment, the human IgG heavy chain constant region is isotype IgG1.In one embodiment, the human IgG heavy chain constant region is isotypeIgG4. In various embodiments, the bispecific antibody comprises achimeric hinge that reduces Fcγ receptor binding relative to a wild-typehinge of the same isotype.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain (HC) and a light chain (LC)amino acid sequence pair (HC/LC) comprising any of the HC amino acidsequences listed in Table 1 paired with any of the LC amino acidsequences listed in Table 1. According to certain embodiments, thepresent invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HC/LC amino acid sequence pair contained withinany of the exemplary anti-FcεR1α antibodies listed in Table 1. Incertain embodiments, the HC/LC amino acid sequence pair is selected fromthe group consisting of SEQ ID NOs: 34/40, 36/40, and 38/40.

The present invention also provides bispecific antibodies, orantigen-binding fragments thereof comprising a first heavy chain, asecond heavy chain and a common light chain comprising any of the HC orLC amino acid sequences listed in Table 7. In certain embodiments, thebispecific antibodies comprise a first HC comprising an amino acidsequence of SEQ ID NO: 56; a second HC comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 50, 52 and 54; and acommon light chain comprising the amino acid sequence of SEQ ID NO: 40.

In one aspect, the invention provides a pharmaceutical compositioncomprising an anti-FcεR1α antigen-binding molecule oranti-FcεR1α/anti-CD3 bispecific antigen-binding molecule and apharmaceutically acceptable carrier or diluent. The invention furtherprovides a method for treating an FcεR1α-related disease, allergy or anIgE-related disease in a subject, the method comprising administering tothe subject the pharmaceutical composition comprising an anti-FcεR1αantigen-binding molecule or anti-FcεR1α/anti-CD3 bispecificantigen-binding molecule and a pharmaceutically acceptable carrier ordiluent. In some embodiments, the allergy or other IgE-related diseasesare selected from the group consisting of allergic asthma, allergicrhinitis, hay fever, anaphylaxis, atopic dermatitis, chronic urticarial,food allergy, perennial allergy, drug allergy, and pollen allergy. Inone embodiment, the allery is severe allergy. In some cases, the allergyleads to anaphylaxis. In certain embodiments, the FcεR1α-related diseasecomprises severe allergy, mast cell activation disorder or mastocytosis.In certain embodiments, the method for treating allergy comprisesadministering to the subject the pharmaceutical composition comprisingan anti-FcεR1α antigen-binding molecule or anti-FcεR1α/anti-CD3bispecific antigen-binding molecule at a certain dose, as describedelsewhere herein.

In another aspect, the present invention provides nucleic acid moleculesencoding any of the HCVR, LCVR or CDR sequences of the anti-FcεR1α, andanti-CD3/anti-FcεR1α bispecific antigen-binding molecules disclosedherein, including nucleic acid molecules comprising the polynucleotidesequences as set forth in Tables 2, and 4 herein, as well as nucleicacid molecules comprising two or more of the polynucleotide sequences asset forth in Tables 2, and 4 in any functional combination orarrangement thereof. Recombinant expression vectors carrying the nucleicacids of the invention, and host cells into which such vectors have beenintroduced, are also encompassed by the invention, as are methods ofproducing the antibodies by culturing the host cells under conditionspermitting production of the antibodies, and recovering the antibodiesproduced.

The present invention provides nucleic acid molecules encoding any ofthe heavy chain amino acid sequences listed in Table 7. The presentinvention also provides nucleic acid molecules encoding any of the lightchain amino acid sequences listed in Table 7.

The present invention includes anti-CD3/anti-FcεR1α bispecificantigen-binding molecules wherein any of the aforementionedantigen-binding domains that specifically bind CD3 are combined,connected or otherwise associated with any of the aforementionedantigen-binding domains that specifically bind FcεR1α to form abispecific antigen-binding molecule that binds CD3 and FcεR1α.

The present invention includes anti-CD3/anti-FcεR1α bispecificantigen-binding molecules having a modified glycosylation pattern. Insome applications, modification to remove undesirable glycosylationsites may be useful, or an antibody lacking a fructose moiety present onthe oligosaccharide chain, for example, to increase antibody dependentcellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, modification of galactosylation canbe made in order to modify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising an anti-CD3/anti-FcεR1α bispecific antigen-binding moleculeas disclosed herein and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition which is acombination of an anti-CD3/anti-FcεR1α bispecific antigen-bindingmolecule and a second therapeutic agent. In one embodiment, the secondtherapeutic agent is any agent that is advantageously combined with ananti-CD3/anti-FcεR1α bispecific antigen-binding molecule. Exemplaryagents that may be advantageously combined with an anti-CD3/anti-FcεR1αbispecific antigen-binding molecule are discussed in detail elsewhereherein.

In yet another aspect, the invention provides therapeutic methods fortargeting/ablating cells expressing FcεR1α using an anti-CD3/anti-FcεR1αbispecific antigen-binding molecule of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising ananti-CD3/anti-FcεR1α bispecific antigen-binding molecule of theinvention to a subject in need thereof. The antibody or fragment thereofmay be administered sub-cutaneously, intravenously, intradermally,intraperitoneally, orally or intramuscularly. In certain embodiments, anantibody of the invention is administered at a dose of about 0.001 mg/kgbody weight to about 200 mg/kg body weight of the subject. In certainembodiments, an antibody of the invention is administered at a dosecomprising between 1 mg to 2500 mg of the antibody to a subject in needthereof.

The present invention also includes the use of an anti-CD3/anti-FcεR1αbispecific antigen-binding molecule of the invention in the manufactureof a medicament for the treatment of a disease or disorder related to orcaused by FcεR1α-expressing cells.

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expression “CD3,” as used herein, refers to an antigen which isexpressed on T cells as part of the multimolecular T cell receptor (TCR)and which consists of a homodimer or heterodimer formed from theassociation of two of four receptor chains: CD3-epsilon, CD3-delta,CD3-zeta, and CD3-gamma. All references to proteins, polypeptides andprotein fragments herein are intended to refer to the human version ofthe respective protein, polypeptide or protein fragment unlessexplicitly specified as being from a non-human species. Thus, theexpression “CD3” means human CD3 unless specified as being from anon-human species, e.g., “mouse CD3,” “monkey CD3,” etc. HumanCD3-epsilon comprises the amino acid sequence set forth as SEQ ID NO:59; human CD3-delta comprises the amino acid sequence set forth as SEQID NO: 60; CD3-zeta comprises the amino acid sequence set forth as SEQID NO: 61; and CD3-gamma comprises the amino acid sequence set forth asSEQ ID NO: 62.

As used herein, “an antibody that binds CD3” or an “anti-CD3 antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize a single CD3 subunit (e.g., epsilon, delta, gammaor zeta), as well as antibodies and antigen-binding fragments thereofthat specifically recognize a dimeric complex of two CD3 subunits (e.g.,gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The antibodiesand antigen-binding fragments of the present invention may bind solubleCD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3proteins as well as recombinant CD3 protein variants such as, e.g.,monomeric and dimeric CD3 constructs, that lack a transmembrane domainor are otherwise unassociated with a cell membrane.

As used herein, the expression “cell surface-expressed CD3” means one ormore CD3 protein(s) that is/are expressed on the surface of a cell invitro or in vivo, such that at least a portion of a CD3 protein isexposed to the extracellular side of the cell membrane and is accessibleto an antigen-binding portion of an antibody. “Cell surface-expressedCD3” includes CD3 proteins contained within the context of a functionalT cell receptor in the membrane of a cell. The expression “cellsurface-expressed CD3” includes CD3 protein expressed as part of ahomodimer or heterodimer on the surface of a cell (e.g., gamma/epsilon,delta/epsilon, and zeta/zeta CD3 dimers). The expression, “cellsurface-expressed CD3” also includes a CD3 chain (e.g., CD3-epsilon,CD3-delta or CD3-gamma) that is expressed by itself, without other CD3chain types, on the surface of a cell. A “cell surface-expressed CD3”can comprise or consist of a CD3 protein expressed on the surface of acell which normally expresses CD3 protein. Alternatively, “cellsurface-expressed CD3” can comprise or consist of CD3 protein expressedon the surface of a cell that normally does not express human CD3 on itssurface but has been artificially engineered to express CD3 on itssurface.

The expression “FcεR1α,” as used herein, refers to an α-chain of thehigh affinity Fc receptor (FcεR1) for IgE. FcεR1α is responsible for thebinding of IgE to FcεR1. FcεR1α is expressed in mast cells, basophils,monocytes, macrophages, mDCs, pDCs, Langerhans cells, eosinophils andplatelets. The amino acid sequence of human FcεR1α is set forth as SEQID NO: 63. The term “FcεR1α” includes recombinant FcεR1α protein or afragment thereof. The term also encompasses FcεR1α protein or a fragmentthereof coupled to, for example, histidine tag, mouse or human Fc, or asignal sequence such as ROR1 (for example, SEQ ID NOs: 57 or 58).

As used herein, “an antibody that binds FcεR1α” or an “anti-FcεR1αantibody” includes antibodies and antigen-binding fragments thereof thatspecifically recognize FcεR1α.

As used herein, the term “disease or disorder associated with expressionof FcεR1α” includes any disease or disorder in which inhibition ofexpression and/or activity (e.g., signaling) of FcεR1α and/or ablationof cells expressing FcεR1α is expected to alleviate symptoms and/orprogression of the disorder. For example, such diseases and disordersinclude, but are not limited to mast cell activation disorders,mastocytosis, and allergy, including but not limited to food allergy,pollen allergy, pet dander allergy, etc.

The term “allergy,” as used herein, refers to a condition caused byhypersensitivity of the immune system to a substance (allergen) in theenvironment. Allergies include, but are not limited to allergic asthma,hay fever, atopic dermatitis, chronic urticaria, food allergy, petdander allergy, and pollen allergy. Symptoms of allergies may include,but are not limited to urticaria (e.g., hives), angioedema, rhinitis,asthma, vomiting, sneezing, runny nose, shortness of breath, sinusinflammation, watery eyes, wheezing, bronchospasm, reduced peakexpiratory flow (PEF), gastrointestinal distress, flushing, swollenlips, swollen tongue, reduced blood pressure, anaphylaxis, and organdysfunction/failure. In one embodiment, the allergy is an anaphylacticallergy, which is a severe form of allergy that may cause death.Symptoms of anaphylaxis may include, but are not limited to rashes,throat or tongue swelling, airway swelling, shortness of breath,vomiting, lightheadedness, low blood pressure, etc.

The term “allergen,” as used herein, includes any substance, chemical,particle or composition which is capable of stimulating an allergicresponse in a susceptible individual. Allergens may be contained withinor derived from a food item such as, e.g., dairy products (e.g., cow'smilk), egg, celery, sesame, wheat, soy, fish, shellfish, sugars (e.g.,sugars present on meat such as alpha-galactose), peanuts, other legumes(e.g., beans, peas, soybeans, etc.), and tree nuts. Alternatively, anallergen may be contained within or derived from a non-food item suchas, e.g., dust (e.g., containing dust mite), pollen, insect venom (e.g.,venom of bees, wasps, mosquitos, fire ants, etc.), mold, animal fur,animal dander, wool, latex, metals (e.g., nickel), household cleaners,detergents, medication, cosmetics (e.g., perfumes, etc.), drugs (e.g.,penicillin, sulfonamides, salicylate, etc.), therapeutic monoclonalantibodies (e.g., cetuximab), ragweed, grass and birch. Exemplary pollenallergens include, e.g., tree pollens such as birch pollen, cedarpollen, oak pollen, alder pollen, hornbeam pollen, aesculus pollen,willow pollen, poplar pollen, plantanus pollen, tilia pollen, oleapollen, Ashe juniper pollen, and Alstonia scholaris pollen.

The term “antigen-binding molecule” includes antibodies andantigen-binding fragments of antibodies, including, e.g., bispecificantibodies.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., FcεR1α or CD3). The term “antibody” includesimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. In different embodiments of the invention, the FRs of theanti-FcεR1α antibody or anti-CD3 antibody (or antigen-binding portionthereof) may be identical to the human germline sequences, or may benaturally or artificially modified. An amino acid consensus sequence maybe defined based on a side-by-side analysis of two or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (iX) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et aL. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments of the invention, the anti-FcεR1α monospecificantibodies or anti-FcεR1α/anti-CD3 bispecific antibodies of theinvention are human antibodies. The term “human antibody”, as usedherein, is intended to include antibodies having variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The present invention also includes one-arm antibodies that bind FcεR1α.As used herein, a “one-arm antibody” means an antigen-binding moleculecomprising a single antibody heavy chain and a single antibody lightchain. The one-arm antibodies of the present invention may comprise anyof the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1.

The anti-FcεR1α or anti-FcεR1α/anti-CD3 antibodies disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes antibodies,and antigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-FcεR1α or anti-FcεR1α/anti-CD3antibodies comprising variants of any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes anti-FcεR1αor anti-FcεR1α/anti-CD3 antibodies having HCVR, LCVR, and/or CDR aminoacid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 orfewer, etc. conservative amino acid substitutions relative to any of theHCVR, LCVR, and/or CDR amino acid sequences set forth in Tables 1 and 3herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Germline Mutations

The anti-FcεR1α and/or anti-CD3 antibodies disclosed herein comprise oneor more amino acid substitutions, insertions and/or deletions in theframework and/or CDR regions of the heavy chain variable domains ascompared to the corresponding germline sequences from which theantibodies were derived.

The present invention also includes antibodies, and antigen-bindingfragments thereof, which are derived from any of the amino acidsequences disclosed herein, wherein one or more amino acids within oneor more framework and/or CDR regions are mutated to the correspondingresidue(s) of the germline sequence from which the antibody was derived,or to the corresponding residue(s) of another human germline sequence,or to a conservative amino acid substitution of the correspondinggermline residue(s) (such sequence changes are referred to hereincollectively as “germline mutations”), and having desired bindingproperties to an FcεR1α or CD3 antigen, for example, weak or nodetectable binding of anti-CD3 antibodies to CD3. Several such exemplaryantibodies that recognize FcεR1α are described in Table 1. Several suchexemplary antibodies that recognize CD3 are described in Table 3.

Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be tested for one or more desired properties such as, improvedbinding specificity, weak or reduced binding affinity, improved orenhanced pharmacokinetic properties, reduced immunogenicity, etc.Antibodies and antigen-binding fragments obtained in this general mannergiven the guidance of the present disclosure are encompassed within thepresent invention.

The present invention also includes anti-FcεR1α antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-CD3 antibodies having HCVR,LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 orfewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences set forth in Table 1 herein. The antibodies and bispecificantigen-binding molecules of the present invention comprise one or moreamino acid substitutions, insertions and/or deletions in the frameworkand/or CDR regions of the heavy and light chain variable domains ascompared to the corresponding germline sequences from which theindividual antigen-binding domains were derived, while maintaining orimproving the desired binding to FcεR1α or CD3, for example, weak or nodetectable binding of anti-CD3 antibodies to CD3 antigen. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein, i.e.the amino acid substitution maintains or improves the desired bindingaffinity in the case of anti-FcεR1α and/or anti-CD3 binding molecules,for example, weak to no detectable binding or anti-CD3 antibodies to CD3antigen. Examples of groups of amino acids that have side chains withsimilar chemical properties include (1) aliphatic side chains: glycine,alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl sidechains: serine and threonine; (3) amide-containing side chains:asparagine and glutamine; (4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, andhistidine; (6) acidic side chains: aspartate and glutamate, and (7)sulfur-containing side chains are cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443-1445. A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

The present invention also includes antigen-binding molecules comprisingan antigen-binding domain with an HCVR and/or CDR amino acid sequencethat is substantially identical to any of the HCVR and/or CDR amino acidsequences disclosed herein, while maintaining or improving the desiredproperty to FcεR1α and/or CD3 antigen. The term “substantial identity”or “substantially identical,” when referring to an amino acid sequencemeans that two amino acid sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at least95% sequence identity, even more preferably at least 98% or 99% sequenceidentity. Preferably, residue positions which are not identical differby conservative amino acid substitutions. In cases where two or moreamino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et aL. (1990) J. Mol. Biol. 215:403-410 and Altschul etaL.(1997) Nucleic Acids Res. 25:3389-402.

Once obtained, antigen-binding domains that contain one or more germlinemutations are tested for decreased binding affinity utilizing one ormore in vitro assays. Although antibodies that recognize a particularantigen are typically screened for their purpose by testing for high(i.e. strong) binding affinity to the antigen, the antibodies of thepresent invention exhibit weak binding or no detectable binding.Bispecific antigen-binding molecules comprising one or moreantigen-binding domains obtained in this general manner are alsoencompassed within the present invention and are found to beadvantageous as avidity-driven allergy therapies.

Unexpected benefits, for example, improved pharmacokinetic propertiesand low toxicity to the patient may be realized from the methodsdescribed herein.

Binding Properties of the Antibodies

As used herein, the term “binding” in the context of the binding of anantibody, immunoglobulin, antibody-binding fragment, or Fc-containingprotein to either, e.g., a predetermined antigen, such as a cell surfaceprotein or fragment thereof, typically refers to an interaction orassociation between a minimum of two entities or molecular structures,such as an antibody-antigen interaction.

For instance, binding affinity typically corresponds to a K_(D) value ofabout 10⁻⁶ M or less, such as about 10⁻⁷ M or less, such as about 10⁻⁸ Mor less, such as about 10⁻⁹ M or less when determined by, for instance,surface plasmon resonance (SPR) technology in a BIAcore 3000 instrumentusing the antigen as the ligand and the antibody, Ig, antibody-bindingfragment, or Fc-containing protein as the analyte (or antiligand).Cell-based binding strategies, such as fluorescent-activated cellsorting (FACS) binding assays, are also routinely used, and FACS datacorrelates well with other methods such as radioligand competitionbinding and SPR (Benedict, C A, J Immunol Methods. 1997, 201(2):223-31;Geuijen, C A, et al. J Immunol Methods. 2005, 302(1-2):68-77).

Accordingly, the antibody or antigen-binding protein of the inventionbinds to the predetermined antigen or cell surface molecule (receptor)having an affinity corresponding to a K_(D) value that is at leastten-fold lower than its affinity for binding to a non-specific antigen(e.g., BSA, casein). According to the present invention, the affinity ofan antibody corresponding to a K_(D) value that is equal to or less thanten-fold lower than a non-specific antigen may be considerednon-detectable binding, however such an antibody may be paired with asecond antigen binding arm for the production of a bispecific antibodyof the invention.

The term “K_(D)” (M) refers to the dissociation equilibrium constant ofa particular antibody-antigen interaction, or the dissociationequilibrium constant of an antibody or antibody-binding fragment bindingto an antigen. There is an inverse relationship between K_(D) andbinding affinity, therefore the smaller the K_(D) value, the higher,i.e. stronger, the affinity. Thus, the terms “higher affinity” or“stronger affinity” relate to a greater ability to form an interactionand therefore a smaller K_(D) value, and conversely the terms “loweraffinity” or “weaker affinity” relate to a lesser ability to form aninteraction and therefore a larger K_(D) value. In some circumstances, ahigher binding affinity (or K_(D)) of a particular molecule (e.g.antibody) to its interactive partner molecule (e.g. antigen X) comparedto the binding affinity of the molecule (e.g. antibody) to anotherinteractive partner molecule (e.g. antigen Y) may be expressed as abinding affinity ratio determined by dividing the larger K_(D) value(lower, or weaker, affinity) by the smaller K_(D) (higher, or stronger,affinity), for example expressed as 5-fold or 10-fold greater bindingaffinity, as the case may be.

The term “k_(d)” (sec-1 or 1/s) refers to the dissociation rate constantof a particular antibody-antigen interaction, or the dissociation rateconstant of an antibody or antibody-binding fragment. Said value is alsoreferred to as the k_(off) value.

The term “k_(a)” (M−1×sec−1 or 1/M) refers to the association rateconstant of a particular antibody-antigen interaction, or theassociation rate constant of an antibody or antibody-binding fragment.

The term “K_(A)” (M−1 or 1/M) refers to the association equilibriumconstant of a particular antibody-antigen interaction, or theassociation equilibrium constant of an antibody or antibody-bindingfragment. The association equilibrium constant is obtained by dividingthe k_(a) by the k_(d).

The term “EC50” or “EC₅₀” refers to the half maximal effectiveconcentration, which includes the concentration of an antibody thatinduces a response halfway between the baseline and maximum after aspecified exposure time. The EC₅₀ essentially represents theconcentration of an antibody where 50% of its maximal effect isobserved. In certain embodiments, the EC₅₀ value equals theconcentration of an antibody of the invention that gives half-maximalbinding to cells expressing CD3 or allergy related antigen, asdetermined by e.g. a FACS binding assay. Thus, reduced or weaker bindingis observed with an increased EC₅₀, or half maximal effectiveconcentration value.

In one embodiment, decreased binding can be defined as an increased EC₅₀antibody concentration which enables binding to the half-maximal amountof target cells.

In another embodiment, the EC₅₀ value represents the concentration of anantibody of the invention that elicits half-maximal depletion of targetcells by T cell cytotoxic activity. Thus, increased cytotoxic activity(e.g. T cell-mediated basophils killing) is observed with a decreasedEC₅₀, or half maximal effective concentration value.

Bispecific Antigen-Binding Molecules

The antibodies of the present invention may be monospecific,bi-specific, or multispecific.

Multispecific antibodies may be specific for different epitopes of onetarget polypeptide or may contain antigen-binding domains specific formore than one target polypeptide. See, e.g., Tutt et al., 1991, J.Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.The anti-FcεR1α monospecific antibodies or anti-FcεR1α/anti-CD3bispecific antibodies of the present invention can be linked to orco-expressed with another functional molecule, e.g., another peptide orprotein. For example, an antibody or fragment thereof can befunctionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multispecific antibody with a second or additionalbinding specificity.

Use of the expression “anti-CD3 antibody” or “anti-FcεR1α antibody”herein is intended to include both monospecific anti-CD3 or anti-FcεR1αantibodies as well as bispecific antibodies comprising a CD3-binding armand an FcεR1α-binding arm. Thus, the present invention includesbispecific antibodies wherein one arm of an immunoglobulin binds humanCD3, and the other arm of the immunoglobulin is specific for humanFcεR1α. The CD3-binding arm can comprise any of the HCVR/LCVR or CDRamino acid sequences as set forth in Table 3 herein.

In certain embodiments, the CD3-binding arm binds to human CD3 andinduces human T cell activation. In certain embodiments, the CD3-bindingarm binds weakly to human CD3 and induces human T cell activation. Inother embodiments, the CD3-binding arm binds weakly to human CD3 andinduces ablation of mast cells and/or basophils in the context of abispecific or multispecific antibody. In other embodiments, theCD3-binding arm binds or is associated weakly with human CD3, yet thebinding interaction is not detectable by in vitro assays known in theart. The FcεR1α-binding arm can comprise any of the HCVR/LCVR or CDRamino acid sequences as set forth in Table 1 herein.

According to certain exemplary embodiments, the present inventionincludes bispecific antigen-binding molecules that specifically bind CD3and FcεR1α. Such molecules may be referred to herein as, e.g.,“anti-CD3/anti-FcεR1α,” or “anti-CD3×FcεR1α,” or “anti-FcεR1α/anti-CD3,”or “anti-FcεR1α×CD3,” or “CD3×FcεR1α” bispecific molecules, or“FcεR1α×CD3” bispecific molecules, or other similar terminology (e.g.,anti-FcεR1α×anti-CD3).

The term “FcεR1α,” as used herein, refers to the human FcεR1α proteinunless specified as being from a non-human species (e.g., “mouseFcεR1α,” “monkey FcεR1α,” etc.). The human FcεR1α protein has the aminoacid sequence shown in SEQ ID NO: 63.

The aforementioned bispecific antigen-binding molecules thatspecifically bind CD3 and FcεR1α may comprise an anti-CD3antigen-binding molecule which binds to CD3 with a weak binding affinitysuch as exhibiting a K_(D) of greater than about 40 nM, as measured byan in vitro affinity binding assay.

As used herein, the expression “antigen-binding molecule” means aprotein, polypeptide or molecular complex comprising or consisting of atleast one complementarity determining region (CDR) that alone, or incombination with one or more additional CDRs and/or framework regions(FRs), specifically binds to a particular antigen. In certainembodiments, an antigen-binding molecule is an antibody or a fragment ofan antibody, as those terms are defined elsewhere herein.

As used herein, the expression “bispecific antigen-binding molecule”means a protein, polypeptide or molecular complex comprising at least afirst antigen-binding domain and a second antigen-binding domain. Eachantigen-binding domain within the bispecific antigen-binding moleculecomprises at least one CDR that alone, or in combination with one ormore additional CDRs and/or FRs, specifically binds to a particularantigen. In the context of the present invention, the firstantigen-binding domain specifically binds a first antigen (e.g., CD3),and the second antigen-binding domain specifically binds a second,distinct antigen (e.g., FcεR1α).

In certain exemplary embodiments of the present invention, thebispecific antigen-binding molecule is a bispecific antibody. Eachantigen-binding domain of a bispecific antibody comprises a heavy chainvariable domain (HCVR) and a light chain variable domain (LCVR). In thecontext of a bispecific antigen-binding molecule comprising a first anda second antigen-binding domain (e.g., a bispecific antibody), the CDRsof the first antigen-binding domain may be designated with the prefix“A1” and the CDRs of the second antigen-binding domain may be designatedwith the prefix “A2”. Thus, the CDRs of the first antigen-binding domainmay be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and theCDRs of the second antigen-binding domain may be referred to herein asA2-HCDR1, A2-HCDR2, and A2-HCDR3.

The first antigen-binding domain and the second antigen-binding domainmay be directly or indirectly connected to one another to form abispecific antigen-binding molecule of the present invention.Alternatively, the first antigen-binding domain and the secondantigen-binding domain may each be connected to a separate multimerizingdomain. The association of one multimerizing domain with anothermultimerizing domain facilitates the association between the twoantigen-binding domains, thereby forming a bispecific antigen-bindingmolecule. As used herein, a “multimerizing domain” is any macromolecule,protein, polypeptide, peptide, or amino acid that has the ability toassociate with a second multimerizing domain of the same or similarstructure or constitution. For example, a multimerizing domain may be apolypeptide comprising an immunoglobulin C_(H)3 domain. A non-limitingexample of a multimerizing component is an Fc portion of animmunoglobulin (comprising a C_(H)2-C_(H)3 domain), e.g., an Fc domainof an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as wellas any allotype within each isotype group.

Bispecific antigen-binding molecules of the present invention willtypically comprise two multimerizing domains, e.g., two Fc domains thatare each individually part of a separate antibody heavy chain. The firstand second multimerizing domains may be of the same IgG isotype such as,e.g., IgG1/IgG1, IgG2/IgG2, and IgG4/IgG4. Alternatively, the first andsecond multimerizing domains may be of different IgG isotypes such as,e.g., IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In certain embodiments, the multimerizing domain is an Fc fragment or anamino acid sequence of from 1 to about 200 amino acids in lengthcontaining at least one cysteine residue. In other embodiments, themultimerizing domain is a cysteine residue, or a shortcysteine-containing peptide. Other multimerizing domains includepeptides or polypeptides comprising or consisting of a leucine zipper, ahelix-loop motif, or a coiled-coil motif.

Any bispecific antibody format or technology may be used to make thebispecific antigen-binding molecules of the present invention. Forexample, an antibody or fragment thereof having a first antigen bindingspecificity can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmenthaving a second antigen-binding specificity to produce a bispecificantigen-binding molecule. Specific exemplary bispecific formats that canbe used in the context of the present invention include, withoutlimitation, e.g., scFv-based or diabody bispecific formats, IgG-scFvfusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes,common light chain (e.g., common light chain with knobs-into-holes,etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody,IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab² bispecific formats (see,e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein,for a review of the foregoing formats).

In the context of bispecific antigen-binding molecules of the presentinvention, the multimerizing domains, e.g., Fc domains, may comprise oneor more amino acid changes (e.g., insertions, deletions orsubstitutions) as compared to the wild-type, naturally occurring versionof the Fc domain. For example, the invention includes bispecificantigen-binding molecules comprising one or more modifications in the Fcdomain that results in a modified Fc domain having a modified bindinginteraction (e.g., enhanced or diminished) between Fc and FcRn. In oneembodiment, the bispecific antigen-binding molecule comprises amodification in a C_(H)2 or a C_(H)3 region, wherein the modificationincreases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).Non-limiting examples of such Fc modifications include, e.g., amodification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/Dor T); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q orK) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or428; or a modification at position 307 or 308 (e.g., F or P), and 434.In one embodiment, the modification comprises a 428L (e.g., M428L) and434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F(e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y)modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E)modification; a 250Q and 428L modification (e.g., T250Q and M428L); anda 307 and/or 308 modification (e.g., 308F or 308P).

The present invention also includes bispecific antigen-binding moleculescomprising a first C_(H)3 domain and a second Ig C_(H)3 domain, whereinthe first and second Ig C_(H)3 domains differ from one another by atleast one amino acid, and wherein at least one amino acid differencereduces binding of the bispecific antibody to Protein A as compared to abi-specific antibody lacking the amino acid difference. In oneembodiment, the first Ig C_(H)3 domain binds Protein A and the second IgC_(H)3 domain contains a mutation that reduces or abolishes Protein Abinding such as an H95R modification (by IMGT exon numbering; H435R byEU numbering). The second C_(H)3 may further comprise a Y96Fmodification (by IMGT; Y436F by EU). See, for example, U.S. Pat. No.8,586,713. Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies.

In certain embodiments, the Fc domain may be chimeric, combining Fcsequences derived from more than one immunoglobulin isotype. Forexample, a chimeric Fc domain can comprise part or all of a C_(H)2sequence derived from a human IgG1, human IgG2 or human IgG4 C_(H)2region, and part or all of a C_(H)3 sequence derived from a human IgG1,human IgG2 or human IgG4. A chimeric Fc domain can also contain achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” sequence, derived from a human IgG1, a human IgG2 or ahuman IgG4 hinge region, combined with a “lower hinge” sequence, derivedfrom a human IgG1, a human IgG2 or a human IgG4 hinge region. Aparticular example of a chimeric Fc domain that can be included in anyof the antigen-binding molecules set forth herein comprises, from N- toC-terminus: [IgG4 C_(H)1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can beincluded in any of the antigen-binding molecules set forth hereincomprises, from N- to C-terminus: [IgG1 C_(H)1]-[IgG1 upper hinge]-[IgG2lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimericFc domains that can be included in any of the antigen-binding moleculesof the present invention are described in US Publication 2014/0243504,published Aug. 28, 2014, which is herein incorporated in its entirety.Chimeric Fc domains having these general structural arrangements, andvariants thereof, can have altered Fc receptor binding, which in turnaffects Fc effector function.

In certain embodiments, the invention provides an antibody heavy chainwherein the heavy chain constant region (CH) region comprises an aminoacid sequence at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identical to any one of SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, or SEQ ID NO: 56. In some embodiments, the heavy chain constantregion (CH) region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQID NO: 56.

Sequence Variants

The antibodies and bispecific antigen-binding molecules of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the individual antigen-binding domains werederived. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. Theantigen-binding molecules of the present invention may compriseantigen-binding domains which are derived from any of the exemplaryamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antigen-binding domain wasoriginally derived. In other embodiments, only certain residues aremutated back to the original germline sequence, e.g., only the mutatedresidues found within the first 8 amino acids of FR1 or within the last8 amino acids of FR4, or only the mutated residues found within CDR1,CDR2 or CDR3. In other embodiments, one or more of the framework and/orCDR residue(s) are mutated to the corresponding residue(s) of adifferent germline sequence (i.e., a germline sequence that is differentfrom the germline sequence from which the antigen-binding domain wasoriginally derived). Furthermore, the antigen-binding domains maycontain any combination of two or more germline mutations within theframework and/or CDR regions, e.g., wherein certain individual residuesare mutated to the corresponding residue of a particular germlinesequence while certain other residues that differ from the originalgermline sequence are maintained or are mutated to the correspondingresidue of a different germline sequence. Once obtained, antigen-bindingdomains that contain one or more germline mutations can be easily testedfor one or more desired property such as, improved binding specificity,increased binding affinity, improved or enhanced antagonistic oragonistic biological properties (as the case may be), reducedimmunogenicity, etc. Bispecific antigen-binding molecules comprising oneor more antigen-binding domains obtained in this general manner areencompassed within the present invention.

The present invention also includes antigen-binding molecules whereinone or both antigen-binding domains comprise variants of any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein having oneor more conservative substitutions. For example, the present inventionincludes antigen-binding molecules comprising an antigen-binding domainhaving HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 orfewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein. A “conservative amino acid substitution” isone in which an amino acid residue is substituted by another amino acidresidue having a side chain (R group) with similar chemical properties(e.g., charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. Examples of groups of amino acids that have side chains withsimilar chemical properties include (1) aliphatic side chains: glycine,alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl sidechains: serine and threonine; (3) amide-containing side chains:asparagine and glutamine; (4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, andhistidine; (6) acidic side chains: aspartate and glutamate, and (7)sulfur-containing side chains are cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443-1445, herein incorporated by reference.A “moderately conservative” replacement is any change having anonnegative value in the PAM250 log-likelihood matrix.

The present invention also includes antigen-binding molecules comprisingan antigen-binding domain with an HCVR, LCVR, and/or CDR amino acidsequence that is substantially identical to any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein. The term “substantialidentity” or “substantially identical,” when referring to an amino acidsequence means that two amino acid sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 95% sequence identity, even more preferably at least 98% or 99%sequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions. In cases where two ormore amino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, herein incorporated by reference.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

pH-Dependent Binding

The present invention includes anti-FcεR1α antibodies, andanti-CD3/anti-FcεR1α bispecific antigen-binding molecules, withpH-dependent binding characteristics. For example, an anti-FcεR1αantibody of the present invention may exhibit reduced binding to FcεR1αat acidic pH as compared to neutral pH. Alternatively, anti-FcεR1αantibodies of the invention may exhibit enhanced binding to FcεR1α atacidic pH as compared to neutral pH. The expression “acidic pH” includespH values less than about 6.2, e.g., about 6.0, 5.95, 5.9, 5.85, 5.8,5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15,5.1, 5.05, 5.0, or less. As used herein, the expression “neutral pH”means a pH of about 7.0 to about 7.4. The expression “neutral pH”includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35,and 7.4.

In certain instances, “reduced binding . . . at acidic pH as compared toneutral pH” is expressed in terms of a ratio of the K_(D) value of theantibody binding to its antigen at acidic pH to the K_(D) value of theantibody binding to its antigen at neutral pH (or vice versa). Forexample, an antibody or antigen-binding fragment thereof may be regardedas exhibiting “reduced binding to FcεR1α at acidic pH as compared toneutral pH” for purposes of the present invention if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0,100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-FcεR1αantibodies, and anti-CD3/anti-FcεR1α bispecific antigen-bindingmolecules, are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes antibodies comprising a mutation in theC_(H)2 or a C_(H)3 region of the Fc domain, wherein the mutation(s)increases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Suchmutations may result in an increase in serum half-life of the antibodywhen administered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or amodification at position 250 and/or 428; or a modification at position307 and/or 308 (e.g., F or P), and 434. In one embodiment, themodification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); a 307 modification and/ora 308 modification (e.g., 308F or 308P).

For example, the present invention includes anti-FcεR1α antibodies, andanti-CD3/anti-FcεR1α bispecific antigen-binding molecules, comprising anFc domain comprising one or more pairs or groups of mutations selectedfrom the group consisting of: 2500 and 248L (e.g., T250Q and M248L);252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g.,M428L and N434S); and 433K and 434F (e.g., H433K and N434F). Allpossible combinations of the foregoing Fc domain mutations, and othermutations within the antibody variable domains disclosed herein, arecontemplated within the scope of the present invention.

Biological Characteristics of the Antibodies and BispecificAntigen-Binding Molecules

According to certain embodiments, the present invention includesantibodies and antigen-binding fragments of antibodies that bind humanFcεR1α (e.g., at 25° C.) with a K_(D) of less than about 303 nM or bindcynomolgus FcεR1α (e.g., at 25° C.) with a K_(D) of less than about 467nM as measured by surface plasmon resonance, e.g., using an assay formatas defined in Example 3 herein. In certain embodiments, the antibodiesor antigen-binding fragments of the present invention bind human orcynomolgus FcεR1α with a K_(D) of less than about 400 nM, less thanabout 500 nM, less than about 450 nM, less than about 400 nM, less thanabout 350 nM, less than about 300 nM, less than about 250 nM, less thanabout 200 nM, less than about 150 nM, or less than about 100 nM, asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Example 3 herein (e.g., mAb-capture or antigen-captureformat), or a substantially similar assay. The present inventionincludes bispecific antigen-binding molecules (e.g., bispecificantibodies which bind human or cynomolgus FcεR1α with a K_(D) of lessthan about 467 nM, as measured by surface plasmon resonance, e.g., usingan assay format as defined in Example 3 herein (e.g., mAb-capture orantigen-capture format), or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind human FcεR1α with a dissociative half-life(t½) of greater than about 0.2 minute or greater than about 0.5 minutesor bind cynomolgus FcεR1α with a dissociative half-life (t½) of greaterthan about 0.3 minute or greater than about 0.6 minute as measured bysurface plasmon resonance at 25° C., e.g., using an assay format asdefined in Example 3 herein, or a substantially similar assay. Thepresent invention includes bispecific antigen-binding molecules (e.g.,bispecific antibodies) which bind human or cynomolgus FcεR1α with aK_(D) of greater than about 0.54 minutes or greater than about 1.1minutes as measured by surface plasmon resonance at 25° C., e.g., usingan assay format as defined in Example 3 herein, or a substantiallysimilar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof which bind specifically to human cell lines whichexpress human or cynomolgus FcεR1α (e.g., HEK293 cells engineered toexpress human or cynomolgus FcεR1α), as determined by a flowcytometry-based detection assay as set forth in Example 4 or asubstantially similar assay.

The present invention also includes anti-CD3/anti-FcεR1α bispecificantigen-binding molecules which exhibit one or more characteristicsselected from the group consisting of: (a) binding to FcεR1α expressedon cell surface in the absence or presence of IgE (see, e.g., Example4); (b) activating human CD3 signaling in the presence of FcεR1αexpressing cells (see, e.g., Example 5); (c) inducing T-cell mediatedapoptosis of FcεR1α expressing cells in vitro (see, e.g., Example 6);(d) inducing T-cell mediated killing of basophils in a peripheral bloodmononuclear cell (PBMC) population in vitro (see, e.g. Example 6); (e)blocking allergen induced mast cell degranulation (e.g., anaphylaxis) inmice expressing human FcεR1α(see, e.g., Example 7); and (f) depletingsplenic basophils in mice expressing human FcεR1α(see, e.g., Example 7).

The present invention includes antibodies and antigen-binding fragmentsthereof that bind human CD3 with high affinity. The present inventionalso includes antibodies and antigen-binding fragments thereof that bindhuman CD3 with medium or low affinity, depending on the therapeuticcontext and particular targeting properties that are desired. Thepresent invention also includes antibodies and antigen-binding fragmentsthereof that bind human CD3 with no measureable affinity. For example,in the context of a bispecific antigen-binding molecule, wherein one armbinds CD3 and another arm binds a target antigen (e.g., FcεR1α), it maybe desirable for the target antigen-binding arm to bind the targetantigen with high affinity while the anti-CD3 arm binds CD3 with onlymoderate or low affinity or no affinity. In this manner, preferentialtargeting of the antigen-binding molecule to cells expressing the targetantigen may be achieved while avoiding general/untargeted CD3 bindingand the consequent adverse side effects associated therewith.

The present invention includes bispecific antigen-binding molecules(e.g., bispecific antibodies) which are capable of simultaneouslybinding to human CD3 and a human FcεR1α. The binding arm that interactswith cells that express CD3 may have weak to no detectable binding asmeasured in a suitable in vitro binding assay. The extent to which abispecific antigen-binding molecule binds cells that express CD3 and/orFcεR1α can be assessed by fluorescence activated cell sorting (FACS), asillustrated in Example 4 herein.

For example, the present invention includes antibodies, antigen-bindingfragments, and bispecific antibodies thereof which specifically bindhuman T-cell lines which express CD3 but do not express FcεR1α, primateT-cells (e.g., cynomolgus peripheral blood mononuclear cells [PBMCs]),and/or FcεR1α-expressing cells.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind human CD3 with weak (i.e.low) or even no detectable affinity.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind monkey (cynomolgus) CD3 withweak (i.e. low) or even no detectable affinity.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind human CD3 and induce T cellactivation.

The present invention includes anti-CD3/anti-FcεR1α bispecificantigen-binding molecules which are capable of inhibiting allergicresponse and/or depleting FcεR1α-expressing cells in a subject (see,e.g., Example 7, in a passive cutaneous anaphylaxis (PCA) or a flowcytometry-based assay, or substantially similar assays). For example,according to certain embodiments, anti-CD3/anti-FcεR1α bispecificantigen-binding molecules are provided, wherein a single administrationof 25 mg/kg of the bispecific antigen-binding molecule to a subjectcauses a reduction in the number of FcεR1α-expressing cells in thesubject (e.g., the number of splenic basophils is significantlyreduced).

Epitope Mapping and Related Technologies

The epitope on CD3 and/or FcεR1α to which the antigen-binding moleculesof the present invention bind may consist of a single contiguoussequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more) amino acids of a CD3 or FcεR1α protein.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) of CD3 or FcεR1α. The antibodiesof the invention may interact with amino acids contained within a singleCD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma), or may interactwith amino acids on two or more different CD3 chains. The term“epitope,” as used herein, refers to an antigenic determinant thatinteracts with a specific antigen binding site in the variable region ofan antibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstances, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antigen-binding domain of an antibody“interacts with one or more amino acids” within a polypeptide orprotein. Exemplary techniques include, e.g., routine cross-blockingassay such as that described Antibodies, Harlow and Lane (Cold SpringHarbor Press, Cold Spring Harbor, NY), alanine scanning mutationalanalysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol248:443-463), and peptide cleavage analysis. In addition, methods suchas epitope excision, epitope extraction and chemical modification ofantigens can be employed (Tomer, 2000, Protein Science 9:487-496).Another method that can be used to identify the amino acids within apolypeptide with which an antigen-binding domain of an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-raycrystallography of the antigen/antibody complex may also be used forepitope mapping purposes.

The present invention further includes anti-FcεR1α antibodies that bindto the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g. antibodies comprising any of the amino acidsequences as set forth in Table 1 herein). Likewise, the presentinvention also includes anti-FcεR1α antibodies that compete for bindingto FcεR1α with any of the specific exemplary antibodies described herein(e.g. antibodies comprising any of the amino acid sequences as set forthin Table 1 herein).

The present invention also includes bispecific antigen-binding moleculescomprising a first antigen-binding domain that specifically binds humanCD3 and/or cynomolgus CD3 with low or detectable binding affinity, and asecond antigen binding domain that specifically binds human orcynomolgus FcεR1α, wherein the first antigen-binding domain binds to thesame epitope on CD3 as any of the specific exemplary CD3-specificantigen-binding domains described herein, and/or wherein the secondantigen-binding domain binds to the same epitope on FcεR1α as any of thespecific exemplary FcεR1α-specific antigen-binding domains describedherein.

Likewise, the present invention also includes bispecific antigen-bindingmolecules comprising a first antigen-binding domain that specificallybinds human CD3, and a second antigen binding domain that specificallybinds human FcεR1α, wherein the first antigen-binding domain competesfor binding to CD3 with any of the specific exemplary CD3-specificantigen-binding domains described herein, and/or wherein the secondantigen-binding domain competes for binding to FcεR1α with any of thespecific exemplary FcεR1α-specific antigen-binding domains describedherein.

One can easily determine whether a particular antigen-binding molecule(e.g., antibody) or antigen-binding domain thereof binds to the sameepitope as, or competes for binding with, a reference antigen-bindingmolecule of the present invention by using routine methods known in theart. For example, to determine if a test antibody binds to the sameepitope on FcεR1α (or CD3) as a reference bispecific antigen-bindingmolecule of the present invention, the reference bispecific molecule isfirst allowed to bind to an FcεR1α protein (or CD3 protein). Next, theability of a test antibody to bind to the FcεR1α(or CD3) molecule isassessed. If the test antibody is able to bind to FcεR1α(or CD3)following saturation binding with the reference bispecificantigen-binding molecule, it can be concluded that the test antibodybinds to a different epitope of FcεR1α (or CD3) than the referencebispecific antigen-binding molecule. On the other hand, if the testantibody is not able to bind to the FcεR1α(or CD3) molecule followingsaturation binding with the reference bispecific antigen-bindingmolecule, then the test antibody may bind to the same epitope of FcεR1α(or CD3) as the epitope bound by the reference bispecificantigen-binding molecule of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference bispecific antigen-binding molecule or if steric blocking (oranother phenomenon) is responsible for the lack of observed binding.Experiments of this sort can be performed using ELISA, RIA, Biacore,flow cytometry or any other quantitative or qualitative antibody-bindingassay available in the art. In accordance with certain embodiments ofthe present invention, two antigen-binding proteins bind to the same (oroverlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess ofone antigen-binding protein inhibits binding of the other by at least50% but preferably 75%, 90% or even 99% as measured in a competitivebinding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antigen-binding proteins aredeemed to bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantigen-binding protein reduce or eliminate binding of the other. Twoantigen-binding proteins are deemed to have “overlapping epitopes” ifonly a subset of the amino acid mutations that reduce or eliminatebinding of one antigen-binding protein reduce or eliminate binding ofthe other.

To determine if an antibody or antigen-binding domain thereof competesfor binding with a reference antigen-binding molecule, theabove-described binding methodology is performed in two orientations: Ina first orientation, the reference antigen-binding molecule is allowedto bind to an FcεR1α protein (or CD3 protein) under saturatingconditions followed by assessment of binding of the test antibody to theFcεR1α (or CD3) molecule. In a second orientation, the test antibody isallowed to bind to an FcεR1α(or CD3) molecule under saturatingconditions followed by assessment of binding of the referenceantigen-binding molecule to the FcεR1α (or CD3) molecule. If, in bothorientations, only the first (saturating) antigen-binding molecule iscapable of binding to the FcεR1α(or CD3) molecule, then it is concludedthat the test antibody and the reference antigen-binding moleculecompete for binding to FcεR1α (or CD3). As will be appreciated by aperson of ordinary skill in the art, an antibody that competes forbinding with a reference antigen-binding molecule may not necessarilybind to the same epitope as the reference antibody, but may stericallyblock binding of the reference antibody by binding an overlapping oradjacent epitope.

Preparation of Antigen-Binding Domains and Construction of BispecificMolecules

Antigen-binding domains specific for particular antigens can be preparedby any antibody generating technology known in the art. Once obtained,two different antigen-binding domains, specific for two differentantigens (e.g., CD3 and FcεR1α), can be appropriately arranged relativeto one another to produce a bispecific antigen-binding molecule of thepresent invention using routine methods. (A discussion of exemplarybispecific antibody formats that can be used to construct the bispecificantigen-binding molecules of the present invention is provided elsewhereherein). In certain embodiments, one or more of the individualcomponents (e.g., heavy and light chains) of the multispecificantigen-binding molecules of the invention are derived from chimeric,humanized or fully human antibodies. Methods for making such antibodiesare well known in the art. For example, one or more of the heavy and/orlight chains of the bispecific antigen-binding molecules of the presentinvention can be prepared using VELOCIMMUNE™ technology. UsingVELOCIMMUNE™ technology (or any other human antibody generatingtechnology), high affinity chimeric antibodies to a particular antigen(e.g., CD3 or FcεR1α) are initially isolated having a human variableregion and a mouse constant region. The antibodies are characterized andselected for desirable characteristics, including affinity, selectivity,epitope, etc. The mouse constant regions are replaced with a desiredhuman constant region to generate fully human heavy and/or light chainsthat can be incorporated into the bispecific antigen-binding moleculesof the present invention.

Genetically engineered animals may be used to make human bispecificantigen-binding molecules. For example, a genetically modified mouse canbe used which is incapable of rearranging and expressing an endogenousmouse immunoglobulin light chain variable sequence, wherein the mouseexpresses only one or two human light chain variable domains encoded byhuman immunoglobulin sequences operably linked to the mouse kappaconstant gene at the endogenous mouse kappa locus. Such geneticallymodified mice can be used to produce fully human bispecificantigen-binding molecules comprising two different heavy chains thatassociate with an identical light chain that comprises a variable domainderived from one of two different human light chain variable region genesegments. (See, e.g., US 2011/0195454). Fully human refers to anantibody, or antigen-binding fragment or immunoglobulin domain thereof,comprising an amino acid sequence encoded by a DNA derived from a humansequence over the entire length of each polypeptide of the antibody orantigen-binding fragment or immunoglobulin domain thereof. In someinstances, the fully human sequence is derived from a protein endogenousto a human. In other instances, the fully human protein or proteinsequence comprises a chimeric sequence wherein each component sequenceis derived from human sequence. While not being bound by any one theory,chimeric proteins or chimeric sequences are generally designed tominimize the creation of immunogenic epitopes in the junctions ofcomponent sequences, e.g. compared to any wild-type human immunoglobulinregions or domains.

Bioequivalents

The present invention encompasses antigen-binding molecules having aminoacid sequences that vary from those of the exemplary molecules disclosedherein but that retain the ability to bind CD3 and/or FcεR1α. Suchvariant molecules may comprise one or more additions, deletions, orsubstitutions of amino acids when compared to parent sequence, butexhibit biological activity that is essentially equivalent to that ofthe described bispecific antigen-binding molecules.

The present invention includes antigen-binding molecules that arebioequivalent to any of the exemplary antigen-binding molecules setforth herein. Two antigen-binding proteins, or antibodies, areconsidered bioequivalent if, for example, they are pharmaceuticalequivalents or pharmaceutical alternatives whose rate and extent ofabsorption do not show a significant difference when administered at thesame molar dose under similar experimental conditions, either singledoes or multiple dose. Some antigen-binding proteins will be consideredequivalents or pharmaceutical alternatives if they are equivalent in theextent of their absorption but not in their rate of absorption and yetmay be considered bioequivalent because such differences in the rate ofabsorption are intentional and are reflected in the labeling, are notessential to the attainment of effective body drug concentrations on,e.g., chronic use, and are considered medically insignificant for theparticular drug product studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antigen-binding protein.

Bioequivalent variants of the exemplary bispecific antigen-bindingmolecules set forth herein may be constructed by, for example, makingvarious substitutions of residues or sequences or deleting terminal orinternal residues or sequences not needed for biological activity. Forexample, cysteine residues not essential for biological activity can bedeleted or replaced with other amino acids to prevent formation ofunnecessary or incorrect intramolecular disulfide bridges uponrenaturation. In other contexts, bioequivalent antigen-binding proteinsmay include variants of the exemplary bispecific antigen-bindingmolecules set forth herein comprising amino acid changes which modifythe glycosylation characteristics of the molecules, e.g., mutationswhich eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, antigen-bindingmolecules are provided which bind to human CD3. Also provided areantigen-binding molecules which bind to human FcεR1α. The presentinvention also includes antigen-binding molecules that bind to human CD3to CD3 from one or more non-human species; and/or antigen-bindingmolecules that bind to human FcεR1α or and to FcεR1α from one or morenon-human species, e.g., cynomolgus.

According to certain exemplary embodiments of the invention,antigen-binding molecules are provided which bind to human CD3 and/orhuman FcεR1α and may bind or not bind, as the case may be, to one ormore of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit,goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus, cynomolgusor chimpanzee CD3 and/or FcεR1α. For example, in a particular exemplaryembodiment of the present invention bispecific antigen-binding moleculesare provided comprising a first antigen-binding domain that binds humanCD3, and a second antigen-binding domain that binds human or cynomolgusFcεR1α.

Therapeutic Formulation and Administration

The present invention provides pharmaceutical compositions comprisingthe antigen-binding molecules of the present invention. Thepharmaceutical compositions of the invention are formulated withsuitable carriers, excipients, and other agents that provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, PA. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad,CA), DNA conjugates, anhydrous absorption pastes, oil-in-water andwater-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antigen-binding molecule administered to a patient may varydepending upon the age and the size of the patient, target disease,conditions, route of administration, and the like. The preferred dose istypically calculated according to body weight or body surface area. Whena bispecific antigen-binding molecule of the present invention is usedfor therapeutic purposes in an adult patient, it may be advantageous tointravenously administer the bispecific antigen-binding molecule of thepresent invention normally at a single dose of about 0.01 to about 50mg/kg body weight, more preferably about 0.1 to about 25, about 1 toabout 25, or about 5 to about 25 mg/kg body weight. Depending on theseverity of the condition, the frequency and the duration of thetreatment can be adjusted. Effective dosages and schedules foradministering a bispecific antigen-binding molecule may be determinedempirically; for example, patient progress can be monitored by periodicassessment, and the dose adjusted accordingly. Moreover, interspeciesscaling of dosages can be performed using well-known methods in the art(e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPENJUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson,Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, andOPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few.Examples of disposable pen delivery devices having applications insubcutaneous delivery of a pharmaceutical composition of the presentinvention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Florida. In yet another embodiment, a controlledrelease system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antigen-Binding Molecules

The present invention includes methods comprising administering to asubject in need thereof a therapeutic composition comprising ananti-FcεR1α antibody or antigen-binding fragment thereof, or abispecific antigen-binding molecule that specifically binds CD3 andFcεR1α. The therapeutic composition can comprise any of the antibodiesor bispecific antigen-binding molecules as disclosed herein and apharmaceutically acceptable carrier or diluent. As used herein, theexpression “a subject in need thereof” means a human or non-human animalthat exhibits one or more symptoms or indicia of an FcεR1α-relateddisease or disorder such as mast cell activation disorder, mastocytosisor an allergy (e.g., a subject suffering from any type of allergies orexhibiting any allergic response), or who otherwise would benefit froman inhibition or reduction in FcεR1α activity or a depletion of FcεR1α+cells (e.g., anaphylaxis).

The antibodies and bispecific antigen-binding molecules of the invention(and therapeutic compositions comprising the same) are useful, interalia, for treating any disease or disorder in which stimulation,activation and/or targeting of an immune response would be beneficial.In particular, the anti-FcεR1α antibodies or the anti-CD3/anti-FcεR1αbispecific antigen-binding molecules of the present invention may beused for the treatment, prevention and/or amelioration of any disease ordisorder associated with or mediated by FcεR1α expression or activity orthe proliferation of FcεR1α+ cells. The mechanism of action by which thetherapeutic methods of the invention are achieved include killing of thecells expressing FcεR1α in the presence of effector cells, for example,by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two ormore of these mechanisms. Cells expressing FcεR1α which can be inhibitedor killed using the bispecific antigen-binding molecules of theinvention include, for example, mast cells and/or basophils.

The antigen-binding molecules of the present invention may be used totreat a disease or disorder associated with IgE or FcεR1α expressionincluding, e.g., mast cell activation disorder (such as mast cellactivation syndrome), mastocytosis, or allergies including allergicasthma, hay fever, anaphylaxis, atopic dermatitis, chronic urticaria,food allergy, and pollen allergy. The allergies may be caused byexposure to one or more allergens, as listed elsewhere herein. Accordingto certain embodiments of the present invention, the anti-FcεR1αantibodies or anti-FcεR1α/anti-CD3 bispecific antibodies are useful fortreating a patient afflicted with severe allergy, including anaphylaxis.According to other related embodiments of the invention, methods areprovided comprising administering an anti-FcεR1α antibody or ananti-CD3/anti-FcεR1α bispecific antigen-binding molecule as disclosedherein to a patient who is afflicted with anaphylaxis.Analytic/diagnostic methods known in the art, such as allergic reactiontest, etc., may be used to ascertain whether a patient suffersanaphylaxis.

According to certain aspects, the present invention provides methods fortreating a disease or disorder associated with FcεR1α expression (e.g.,anaphylaxis) comprising administering one or more of the anti-FcεR1α orbispecific antigen-binding molecules described elsewhere herein to asubject after the subject has been determined to have allergy. Forexample, the present invention includes methods for treating allergycomprising administering an anti-FcεR1α antibody or ananti-CD3/anti-FcεR1α bispecific antigen-binding molecule to a patient 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more afterthe subject has received other therapy (e.g., anti-histamine therapy).

Combination Therapies and Formulations

The present invention provides methods which comprise administering apharmaceutical composition comprising any of the exemplary antibodiesand bispecific antigen-binding molecules described herein in combinationwith one or more additional therapeutic agents.

Exemplary additional therapeutic agents that may be combined with oradministered in combination with an antigen-binding molecule of thepresent invention include, e.g., an IgE antagonist (e.g., an anti-IgEantibody such as omalizumab) or small molecule inhibitor of IgE (e.g.,darpins such as darpin E2_76), an IL-25 inhibitor, an IL-4 inhibitor, anIL-4 receptor inhibitor (e.g., an anti-IL-4R antibody such asdupilumab), an IL-33 inhibitor (e.g., an anti-IL-33 antibody), a plasmacell ablating agent (e.g., a BCMA×CD3 bispecific antibody) and a TSLPinhibitor. In certain embodiments, the plasma cell ablating agent isselected from the group consisting of a B-cell maturation antigen (BCMA)targeting agent, a proteasome inhibitor, a histone deacetylaseinhibitor, a B-cell activating factor (BAFF) inhibitor, and an inhibitorof A proliferation inducing ligand (APRIL; CD256). In one embodiment,the BCMA targeting agent is selected from the group consisting of ananti-BCMA/anti-CD3 bispecific antibody, a chimeric antigen receptoragainst BCMA, and an anti-BCMA antibody conjugated to a cytotoxic drug.

Other agents that may be beneficially administered in combination withthe antigen-binding molecules of the invention include allergy treatmentmedicines, including antihistamines, anti-inflammatory agents,corticoids, epinephrine, a bronchial dilator, a decongestant,leukotriene antagonists, or mast cell inhibitor (e.g., cromolyn sodium).

The present invention also includes therapeutic combinations comprisingany of the antigen-binding molecules mentioned herein and an inhibitorof IgE or FcεR1α, wherein the inhibitor is an aptamer, an antisensemolecule, a ribozyme, an siRNA, a peptibody, a nanobody or an antibodyfragment (e.g., Fab fragment; F(ab′)₂ fragment; Fd fragment; Fvfragment; scFv; dAb fragment; or other engineered molecules, such asdiabodies, triabodies, tetrabodies, minibodies and minimal recognitionunits). The antigen-binding molecules of the invention may also beadministered and/or co-formulated in combination with antivirals,antibiotics, analgesics, corticosteroids and/or NSAIDs.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan antigen-binding molecule of the present invention; (for purposes ofthe present disclosure, such administration regimens are considered theadministration of an antigen-binding molecule “in combination with” anadditional therapeutically active component).

The present invention includes pharmaceutical compositions in which anantigen-binding molecule of the present invention is co-formulated withone or more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an antigen-binding molecule (e.g., an anti-FcεR1α antibody or abispecific antigen-binding molecule that specifically binds FcεR1α andCD3) may be administered to a subject over a defined time course. Themethods according to this aspect of the invention comprise sequentiallyadministering to a subject in need thereof multiple doses of anantigen-binding molecule of the invention. As used herein, “sequentiallyadministering” means that each dose of an antigen-binding molecule isadministered to the subject at a different point in time, e.g., ondifferent days separated by a predetermined interval (e.g., hours, days,weeks or months). The present invention includes methods which comprisesequentially administering to the patient a single initial dose of anantigen-binding molecule, followed by one or more secondary doses of theantigen-binding molecule, and optionally followed by one or moretertiary doses of the antigen-binding molecule.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the antigen-bindingmolecule of the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of theantigen-binding molecule, but generally may differ from one another interms of frequency of administration. In certain embodiments, however,the amount of an antigen-binding molecule contained in the initial,secondary and/or tertiary doses varies from one another (e.g., adjustedup or down as appropriate) during the course of treatment. In certainembodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered atthe beginning of the treatment regimen as “loading doses” followed bysubsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21,21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks afterthe immediately preceding dose. The phrase “the immediately precedingdose,” as used herein, means, in a sequence of multiple administrations,the dose of antigen-binding molecule which is administered to a patientprior to the administration of the very next dose in the sequence withno intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an antigen-binding molecule (e.g., an anti-FcεR1α antibody or abispecific antigen-binding molecule that specifically binds FcεR1α andCD3). For example, in certain embodiments, only a single secondary doseis administered to the patient. In other embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to thepatient. Likewise, in certain embodiments, only a single tertiary doseis administered to the patient. In other embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to thepatient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

In one embodiment, the antigen-binding molecule (e.g., an anti-FcεR1αantibody or a bispecific antigen-binding molecule that specificallybinds FcεR1α and CD3) is administered to a subject as a weight-baseddose. A “weight-based dose” (e.g., a dose in mg/kg) is a dose of theantibody or the antigen-binding fragment thereof or the bispecificantigen-binding molecule that will change depending on the subject'sweight.

In another embodiment, an antibody or the antigen-binding fragmentthereof or a bispecific antigen-binding molecule is administered to asubject as a fixed dose. A “fixed dose” (e.g., a dose in mg) means thatone dose of the antibody or the antigen-binding fragment thereof or thebispecific antigen-binding molecule is used for all subjects regardlessof any specific subject-related factors, such as weight. In oneparticular embodiment, a fixed dose of an antibody or theantigen-binding fragment thereof or a bispecific antigen-bindingmolecule of the invention is based on a predetermined weight or age.

In general, a suitable dose of the antigen binding molecule theinvention can be in the range of about 0.001 to about 200.0 milligramper kilogram body weight of the recipient, generally in the range ofabout 1 to 50 mg per kilogram body weight. For example, the antibody orthe antigen-binding fragment thereof or the bispecific antigen-bindingmolecule can be administered at about 0.1 mg/kg, about 0.5 mg/kg, about1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 5 mg/kg,about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30mg/kg, about 40 mg/kg, about 50 mg/kg per single dose. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In some embodiments, the antigen binding molecule of the invention isadministered as a fixed dose of between about 1 mg to about 2500 mg. Insome embodiments, the antigen binding molecule of the invention isadministered as a fixed dose of about 1 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15mg, 20 mg, 25 mg, about 30 mg, about 50 mg, about 75 mg, about 100 mg,about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about1000 mg, about 1500 mg, about 2000 mg, or about 2500 mg. Values andranges intermediate to the recited values are also intended to be partof this invention.

Diagnostic Uses of the Antibodies

The anti-FcεR1α antibodies of the present invention may also be used todetect and/or measure FcεR1α, or FcεR1α-expressing cells in a sample,e.g., for diagnostic purposes. For example, an anti-FcεR1α antibody, orfragment thereof, may be used to diagnose a condition or diseasecharacterized by aberrant expression (e.g., over-expression,under-expression, lack of expression, etc.) of FcεR1α. Exemplarydiagnostic assays for FcεR1α may comprise, e.g., contacting a sample,obtained from a patient, with an anti-FcεR1α antibody of the invention,wherein the anti-FcεR1α antibody is labeled with a detectable label orreporter molecule. Alternatively, an unlabeled anti-FcεR1α antibody canbe used in diagnostic applications in combination with a secondaryantibody which is itself detectably labeled. The detectable label orreporter molecule can be a radioisotope, such as ³H, ¹⁴C ³²P, ³⁵S, or¹²⁵I; a fluorescent or chemiluminescent moiety such as fluoresceinisothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Anotherexemplary diagnostic use of the anti-FcεR1α antibodies of the inventionincludes ⁸⁹Zr-labeled, such as ⁸⁹Zr-desferrioxamine-labeled, antibodyfor the purpose of noninvasive identification and tracking of mastcells, basophils, or other FcεR1α expressing cells in a subject (e.g.positron emission tomography (PET) imaging). (See, e.g., Tavare, R. etal. Cancer Res. 2016 Jan. 1; 76(1):73-82; and Azad, B B. et al.Oncotarget. 2016 Mar. 15; 7(11):12344-58.) Specific exemplary assaysthat can be used to detect or measure FcεR1α in a sample includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence-activated cell sorting (FACS).

Samples that can be used in FcεR1α diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of FcεR1α protein, orfragments thereof, under normal or pathological conditions. Generally,levels of FcεR1α in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal FcεR1α levels or activity) will be measured to initiallyestablish a baseline, or standard, level of FcεR1α. This baseline levelof FcεR1α can then be compared against the levels of FcεR1α measured insamples obtained from individuals suspected of having an FcεR1α relateddisease (e.g., a subject with allergy) or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1: Generation of Antibodies Generation of Anti-FcεR1α Antibodies

Anti-FcεR1α antibodies were obtained by immunizing a geneticallymodified mouse with a human FcεR1α antigen (e.g., hFcεR1α, SEQ ID NO:63) or by immunizing an engineered mouse comprising DNA encoding humanimmunoglobulin heavy and kappa light chain variable regions with a humanFcεR1α antigen.

Following immunization, splenocytes were harvested from each mouse andeither (1) fused with mouse myeloma cells to preserve their viabilityand form hybridoma cells and screened for FcεR1α specificity, or (2)B-cell sorted (as described in US 2007/0280945A1) using a human FcεR1αfragment as the sorting reagent that binds and identifies reactiveantibodies (antigen-positive B cells).

Chimeric antibodies to FcεR1α were initially isolated having a humanvariable region and a mouse constant region. The antibodies werecharacterized and selected for desirable characteristics, includingaffinity, selectivity, etc. If necessary, mouse constant regions werereplaced with a desired human constant region, for example wild-type ormodified IgG1 or IgG4 constant region, to generate a fully humananti-FcεR1α antibody. While the constant region selected may varyaccording to specific use, high affinity antigen-binding and targetspecificity characteristics reside in the variable region.

Certain biological properties of the exemplary anti-FcεR1α antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Generation of Anti-CD3 Antibodies

Anti-CD3 antibodies were generated as described in WO 2017/053856, whichis herein incorporated by reference. An exemplary anti-CD3 antibody wasselected for the production of bispecific anti-CD3/anti-FcεR1αantibodies in accordance with the present invention. Other anti-CD3antibodies for use in preparing bispecific antibodies in accordance withthe present invention can be found in, e.g., WO 2014/047231.

Certain biological properties of the exemplary anti-CD3 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples herein.

Generation of Bispecific Antibodies that Bind FcεR1α and CD3

The present invention provides bispecific antigen-binding molecules thatbind CD3 and FcεR1α; such bispecific antigen-binding molecules are alsoreferred to herein as “anti-FcεR1α/anti-CD3 or anti-FcεR1α×CD3bispecific molecules.” The anti-FcεR1α portion of theanti-FcεR1α/anti-CD3 bispecific molecule is useful for targeting cellsthat express FcεR1α, and the anti-CD3 portion of the bispecific moleculeis useful for activating T-cells. The simultaneous binding of FcεR1α ona cell and CD3 on a T-cell facilitates directed killing (cell lysis) ofthe targeted FcεR1α expressing cell by the activated T-cell.

Bispecific antibodies comprising an anti-FcεR1α-specific binding domainand an anti-CD3-specific binding domain were constructed using standardmethodologies, wherein the anti-FcεR1α antigen binding domain and theanti-CD3 antigen binding domain each comprise different, distinct HCVRspaired with a common LCVR. In exemplified bispecific antibodies, themolecules were constructed utilizing a heavy chain from an anti-CD3antibody, a heavy chain from an anti-FcεR1α antibody and a common lightchain from the anti-CD3 antibody WO 2017/053856). In other instances,the bispecific antibodies may be constructed utilizing a heavy chainfrom an anti-CD3 antibody, a heavy chain from an anti-FcεR1α antibodyand a light chain from an anti-CD3 antibody or a light chain from ananti-FcεR1α antibody light chain or any other light chain known to bepromiscuous or pair effectively with a variety of heavy chain arms. Theanti-FcεR1α antibodies and the anti-CD3 antibodies, from which anycomponents of the bispecific antibodies are derived, are sometimesreferred to as parental antibodies.

The bispecific antibodies described in the following examples compriseanti-CD3 binding arms; and anti-FcεR1α binding arm. Exemplifiedbispecific antibodies were manufactured having an IgG4 Fc domain(bsAb24919D, bsAb24920D, and bsAb24921 D).

A summary of the component parts of the antigen-binding domains of thevarious anti-FcεR1α×CD3 bispecific antibodies constructed is set forthin Table 5.

Example 2: Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions (HCVR and LCVR), CDRs and heavy chains andlight chains (HC and LC) of selected anti-FcεR1α antibodies of theinvention. The corresponding nucleic acid sequence identifiers are setforth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers SEQ ID NOs: Antibody LCDR2Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 (AAS) LCDR3 HC LC mAb171102 4 6 8 26 28 30 32 34 40 mAb17111 10 12 14 16 26 28 30 32 36 40mAb17112 18 20 22 24 26 28 30 32 38 40 LCDR2 has an amino acid sequenceof Ala Ala Ser (AAS) (SEQ ID NO: 30).

TABLE 2 Nucleic Acid Sequence Identifiers SEQ ID NOs: LCDR2 Antibody(gctgca Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 tcc) LCDR3 HC LCmAb17110 1 3 5 7 25 27 29 31 33 39 mAb17111 9 11 13 15 25 27 29 31 35 39mAb17112 17 19 21 23 25 27 29 31 37 39 LCDR2 has corresponding nucleicacid sequence of gctgcatcc (SEQ ID NO: 29).

Table 3 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions (HCVR and LCVR), CDRs and heavy chain andlight chain (HC and LC) of an exemplary anti-CD3 antibody of theinvention. The corresponding nucleic acid sequence identifiers are setforth in Table 4.PG-3T

TABLE 3 Amino Acid Sequence Identifiers SEQ ID NOs: Antibody LCDR2 LCDRDesignation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 (AAS) 3 HC LC mAb7221G2042 44 46 48 26 28 30 32 56 40

TABLE 4 Nucleic Acid Sequence Identifiers SEQ ID NOs: LCDR2 Antibody(gctgca Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 tcc) LCDR3 HC LCmAb7221G20 41 43 45 47 25 27 29 31 55 39

A summary of the component parts of the various anti-FcεR1α×CD3bispecific antibodies constructed is set forth in Table 5. Tables 6, 7and 8 list the HCVR, LCVR, CDRs and heavy chain and light sequenceidentifiers of the bispecific antibodies.

TABLE 5 Summary of Component Parts of Anti-FcεR1α16 × CD3 BispecificAntibodies Bispecific Anti-FcεR1α Anti-CD3 Antibody Antigen-BindingDomain Antigen-Binding Domain Common Light Chain Identifier Heavy ChainVariable Region Heavy Chain Variable Region Variable Region bsAb24919DmAb17110 mAb7221G20 mAb7221G20 bsAb24920D mAb17111 mAb7221G20 mAb7221G20bsAb24921D mAb17112 mAb7221G20 mAb7221G20

TABLE 6 Amino acid sequences of variable regions and CDRs of bispecificantibodies Anti-FcεR1α antigen- Anti-CD3 antigen- Common Light chainbinding domain binding domain variable region Bispecific SEQ ID NOs. SEQID NOs. SEQ ID NOs Antibody HCDR HCDR HCDR HCDR HCDR LCDR LCDR 2 LCDRIdentifier HCVR HCDR1 2 3 HCVR 1 2 3 LCVR 1 (AAS) 3 bsAb24919D 2 4 6 842 44 46 48 26 28 30 32 bsAb24920D 10 12 14 16 42 44 46 48 26 28 30 32bsAb24921D 18 20 22 24 42 44 46 48 26 28 30 32

TABLE 7 Heavy chain and light chain amino acid sequence identifiers ofbispecific antibodies Bispecific antibody Anti-FcεR1α Anti-CD3 CommonLight Identifier Heavy Chain Heavy Chain Chain bsAb24919D SEQ ID NO: 50SEQ ID NO: 56 SEQ ID NO: 40 bsAb24920D SEQ ID NO: 52 SEQ ID NO: 56 SEQID NO: 40 bsAb24921D SEQ ID NO: 54 SEQ ID NO: 56 SEQ ID NO: 40

TABLE 8 Heavy chain and light chain nucleic acid sequence identifiers ofbispecific antibodies Bispecific antibody Anti-FcεR1α Anti-CD3 CommonLight Identifier Heavy Chain Heavy Chain Chain bsAb24919D SEQ ID NO: 49SEQ ID NO: 55 SEQ ID NO: 39 bsAb24920D SEQ ID NO: 51 SEQ ID NO: 55 SEQID NO: 39 bsAb24921D SEQ ID NO: 53 SEQ ID NO: 55 SEQ ID NO: 39

Example 3: Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-FcεR1α Monospecific andAnti-FcεR1α16×CD3 Bispecific Antibodies

Equilibrium dissociation constants (K_(D)) for human or cynomolgusFcεR1α ectodomain binding to purified anti-FcεR1α monoclonal antibodies(mAbs) and CD3×FcεR1α bispecific antibodies (bsAbs) were determinedusing a real-time surface plasmon resonance biosensor (SPR-Biacore),Biacore 8 k. All binding studies were performed in 10 mM HEPES, 150 mMNaCl, 3 mM EDTA, and 0.05% v/v surfactant Tween-20, pH 7.4 (HBS-ET)running buffer at 25° C. and 37° C.

The Biacore CM4 sensor surface was first derivatized by amine couplingwith a monoclonal mouse anti-human Fc antibody to capture approximately500-900 RUs anti-FcεR1α monoclonal antibodies or anti-CD3×FcεR1αbispecific monoclonal antibodies. 1 RU (response unit) represents 1 pgof protein per mm², as defined by the manufacturer. The ectodomain ofhuman and cynomolgus FcεR1α reagents were expressed with a C-termmyc-myc-6×His tag-hFcεR1α.MMH (SEQ ID NO: 57) and mfFcεR1α.MMH (SEQ IDNO: 58). Different concentrations of FcεR1α reagents were prepared inHBS-ET running buffer (600 nM-7.4 nM; serially diluted by 3-fold) andinjected over anti-human Fc captured anti-FcεR1α monoclonal antibodiesor anti-CD3×FcεR1α bispecific monoclonal antibodies surfaces for 1minute at a flow rate of 30 μL/minute. The dissociation of bound FcεR1αreagents was monitored for 4 minutes in HBS-ET running buffer.Association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by fitting the real-time binding sensorgrams to a 1:1 bindingmodel with mass transport limitation using Biacore 8 k evaluationsoftware. Binding dissociation equilibrium constants (K_(D)) anddissociative half-lives (t½) were calculated from the kinetic rateconstants as:

${{K_{D}(M)} = \frac{k_{d}}{k_{a}}},{{{and}t{1/2}\left( \min \right)} = \frac{\ln(2)}{60*{kd}}}$

Binding kinetics parameters for hFcεR1α.MMH and mfFcεR1α.MMH binding todifferent exemplary anti-FcεR1α monoclonal antibodies or anti-CD3×FcεR1αbispecific monoclonal antibodies of the invention at 25° C. and 37° C.are shown in Table 9 through Table 12.

TABLE 9 Binding Kinetics Parameters of hFcεR1α.MMH Binding toAnti-FcεR1α Monoclonal Antibodies or Anti-CD3 × FcεR1α BispecificMonoclonal Antibodies at 25° C. mAb 600 nM Capture hFcεR1α.MMH k_(a)k_(d) K_(D) t½ mAb Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb17110 609 ± 1.2 130 7.19E+04 9.13E−03 1.27E−07 1.3 mAb17111 567 ± 0.891 7.84E+04 1.65E−02 2.11E−07 0.7 mAb17112 630 ± 0.9 123 8.80E+041.84E−02 2.09E−07 0.63 bsAb24919D 610 ± 0.7 72 6.82E+04 1.04E−021.52E−07 1.1 bsAb24920D 573 ± 1  46 6.59E+04 2.00E−02 3.03E−07 0.6bsAb24921D 607 ± 0.6 69 7.74E+04 2.12E−02 2.74E−07 0.54 Isotype Control545 ± 1.7 −5 NR * NB * NB * NB * mAb NB * indicates that no binding wasobserved under the current experimental conditions.

TABLE 10 Binding Kinetics Parameters of hFcεR1α.MMH Binding toAnti-FcεR1α Monoclonal Antibodies or Anti-CD3 × FcεR1α BispecificMonoclonal Antibodies at 37° C. mAb 600 nM Capture hFcεR1α.MMH k_(a)k_(d) K_(D) t½ mAb Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb17110 745 ± 1.5 93 8.88E+04 3.94E−02 4.44E−07 0.29 mAb17111 745 ± 2.252 8.21E+04 4.83E−02 5.88E−07 0.24 mAb17112 681 ± 0.6 87 1.13E+055.85E−02 5.17E−07 0.20 bsAb24919D 764 ± 1.5 43 7.35E+04 5.88E−028.01E−07 0.20 bsAb24920D 743 ± 3.5 19 6.39E+04 9.91E−02 1.55E−06 0.12bsAb24921D 670 ± 0.6 40 9.56E+04 9.34E−02 9.77E−07 0.12 Isotype Control688 ± 3.5 −2 NB * NB * NB * NB * mAb NB * indicates that no binding wasobserved under the current experimental conditions.

TABLE 11 Binding Kinetics Parameters of mfFcεR1α.MMH Binding toAnti-FcεR1α Monoclonal Antibodies or Anti-CD3 × FcεR1α BispecificMonoclonal Antibodies at 25° C. mAb 600 nM Capture mfFcεR1α.MMH k_(a)k_(d) K_(D) t½ mAb Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb17110 596 ± 0.8 79 6.13E+04 1.36E−02 2.21E−07 0.8 mAb17111 620 ± 0.560 5.55E+04 1.37E−02 2.47E−07 0.8 mAb17112 594 ± 1.3 110 7.13E+041.39E−02 1.95E−07 0.8 bsAb24919D 597 ± 0.8 40 3.96E+04 1.44E−02 3.63E−070.8 bsAb24920D 625 ± 0.8 26 4.17E+04 1.95E−02 4.67E−07 0.6 bsAb24921D563 ± 0.8 60 6.29E+04 1.61E−02 2.56E−07 0.7 Isotype Control 610 ± 1.6 −9NB * NB * NB * NB * mAb NB * indicates that no binding was observedunder the current experimental conditions.

TABLE 12 Binding Kinetics Parameters of mfFcεR1α.MMH Binding toAnti-FcεR1α Monoclonal Antibodies or Anti-CD3 × FcεR1α BispecificMonoclonal Antibodies at 37° C. mAb 600nM Capture mfFcεR1α.MMH k_(a)k_(d) K_(D) t½ mAb Captured Level (RU) Bound (RU) (1/Ms) (1/s) (M) (min)mAb17110 719 ± 3.5 53 5.94E+04 2.54E−02 4.28E−07 0.45 mAb17111 720 ± 3.438 5.17E+04 2.64E−02 5.10E−07 0.44 mAb17112 769 ± 0.7 81 9.94E+043.28E−02 3.30E−07 0.35 bsAb24919D 685 ± 1.7 20 3.86E+04 3.99E−021.03E−06 0.29 bsAb24920D 660 ± 2.7 10 1.33E+04 4.45E−02 3.35E−06 0.26bsAb24921D 749 ± 1.2 37 8.56E+04 4.45E−02 5.20E−07 0.26 Isotype Control775 ± 2.8 −9 NB * NB * NB * NB * mAb NB * indicates that no binding wasobserved under the current experimental conditions.

At 25° C., exemplary anti-FcεR1α monoclonal antibodies oranti-CD3×FcεR1α bispecific monoclonal antibodies of the invention boundto hFcεR1α.MMH with K_(D) values ranging from 127 nM to 303 nM, as shownin Table 9. At 37° C., exemplary anti-FcεR1α monoclonal antibodies oranti-CD3×FcεR1α bispecific monoclonal antibodies of the invention boundto hFcεR1α.MMH with K_(D) values ranging from 444 nM to 1.55 uM, asshown in Table 10.

At 25° C., exemplary anti-FcεR1α monoclonal antibodies oranti-CD3×FcεR1α bispecific monoclonal antibodies of the invention boundto mfFcεR1α.MMH with K_(D) values ranging from 195 nM to 467 nM, asshown in Table 11. At 37° C., exemplary anti-FcεR1α monoclonalantibodies or anti-CD3×FcεR1α bispecific monoclonal antibodies of theinvention bound to mfFcεR1α.MMH with K_(D) values ranging from 330 nM to3.35 uM, as shown in Table 12.

Example 4: Anti-FcεR1α×CD3 Bispecific Antibodies Bind Specifically toExpressed FcεR1α on Jurkat and HEK293 Cells

In order to assess the binding to antigens expressed on cells byanti-FcεR1α monoclonal antibodies and anti-FcεR1α×CD3 bi-specificantibodies, flow cytometry experiment was performed with Jurkat/NFAT-Lucand HEK293/hFcεR1α/hFcεR1β/hFcεR1γ cells. Jurkat/NFAT-Luc cells areJurkat cells engineered to stably express a luciferase reporter underthe transcription control of Nuclear factor of activated T-cells (NFAT)response element. HEK293/hFcεR1α/hFcεR1β/hFcεR1γ cells are HEK293 cellsengineered to stably express human FcεR1α, FcεR1β and FcεR1γ. To testthe binding to monkey (cynomolgus, mf) FcεR1α (amino acids 4-260 ofaccession #XP_005541370.1 with alanine at position 81 changed totryptophan), mfFcεR1α was stably expressed in HEK293 along with humanFcεR1β and FcεR1γ. The resulting cell line, referred to hereafter asHEK293/mf FcεR1α/hFcεR1β/hFcεR1γ was isolated and maintained in DMEMmedium supplemented with 10% FBS, 1×NEAA, 1×Penicillin/Streptomycin/L-Glutamine, 1 g/mL Puromycin, 100 g/mL ofHygromycin B and 500 g/ml of G418 sulfate. The reagents information isas follows: DMEM medium, Irvine Scientific, Cat #CRL-1573; Fetal bovineserum (FBS), Seradigm, Cat #1500-500; 100×Penicillin/Streptomycin/L-Glutamine (Pen/Strep/Glut), Invitrogen, Cat#10378-016; 100× Non-essential amino acids (NEAA), Irvine Scientific,Cat #9034; Geneticin™ Selective Antibiotic (G418 Sulfate), Invitrogen,Cat #11811-098; Hygromycin B, Calbiochem, Cat #400049; Puromycin, Sigma,Cat #P-8833.

For flow cytometry analysis, HEK293, HEK293/hFcεR1α/hFcεR1β/hFcεR1γ andHEK293/mf FcεR1α/hFcεR1β/hFcεR1γ cells were collected after dissociationusing Enzyme Free Dissociation Buffer (Millipore Cat #S-004), and thecells were pre-incubated with or without 70 nM human IgE, for 30 minuteson ice in FACS buffer (PBS, without Ca⁺⁺ and Mg⁺⁺, (Irvine Scientific,Cat #9240) containing 2% FBS). Jurkat/NFAT-luc cells were alsocollected. The antibodies at the concentration of 70 nM were then addedto 1×10⁶ cells/well of each cell type at 4° C. for 30 minutes. Afterincubation with primary antibodies, the cells were stained with 1.3μg/ml of Allophycocyanin (APC) conjugated anti-human IgG secondaryantibody (Jackson ImmunoResearch, Cat #109-136-170) for 30 minutes onice. Cells were fixed using BD CytoFix™ (Becton Dickinson, Cat. #554655)and analyzed on Accuri™ C6 (BD) or CytoFLEX Flow cytometer (BeckmanCoulter). Unstained and secondary antibody alone controls were alsotested for all cell lines and a sample was evaluated for viability usingthe Far Red Fluo viability dye (Thermo Fisher, Cat #L10120) according tothe manufacturer's protocol. The results were analyzed using FlowJosoftware (version 10.0.8, FlowJo) to determine the geometric means offluorescence for viable cells and the binding ratio was calculated withthe mean fluorescence intensity (MFI) of the experimental conditionnormalized by the MFI of the unstained respective cells. The resultswere summarized in Tables 13 and 14.

TABLE 13 Binding of 70 nM of Anti-FcεR1α Antibodies and Anti-FcεR1α ×CD3 Antibodies to HEK293/hFcεR1α/hFcεR1β/hFcεR1γ and Jurkat/NFAT-lucCells Binding Ratio (MFI of Treated/MFI of Unstained)HEK293/hFcεR1α/hFcεR1β/ Jurkat/NFAT- Antibodies HEK293 hFcεR1γ luc IDSpecificity No IgE No IgE 70 nM IgE No IgE bsAb24919D FcεR1α × hCD3 2269 277 48 bsAb24920D FcεR1α × hCD3 4 248 201 48 bsAb24921D FcεR1α ×hCD3 11 253 295 94 mAb17110 FcεR1α 2 171 396 1 mAb17111 FcεR1α 1 136 3691 mAb17112 FcεR1α 8 158 367 1 Human IgG4 Irrelevant 1 1 1 2 Stealthprotein Control Human IgG4 Irrelevant 1 1 1 1 Control protein

TABLE 14 Binding of 70 nM of Anti-FcεR1α Antibodies and Anti- FcεR1α ×CD3ε Antibodies to HEK293/mf FcεR1α/hFcεR1β/hFcεR1γ Cells Binding Ratio(MFI of Treated/MFI of Unstained) HEK293 Antibodies ParentalHEK293/mfFcεR1α/hFcεR1β/hFcεR1γ ID Specificity No IgE No IgE 70 nM IgEbsAb24919D FcεR1α × hCD3 1 166 236 bsAb24920D FcεR1α × hCD3 2 142 169bsAb24921D FcεR1α × hCD3 2 169 284 mAb17110 FcεR1α 1 101 256 mAb17111FcεR1α 1 102 190 mAb17112 FcεR1α 1 134 279 Human IgG4 Irrelevant 1 1 1Stealth Control protein Human IgG4 Irrelevant 1 1 1 Control protein

As shown in Table 13, exemplary anti-FcεR1α×CD3 bispecific antibodiesbsAb24919D, bsAb24920D, and bsAb24921D, showed binding to human FcεR1αexpressed in HEK293/hFcεR1α/hFcεR1β/hFcεR1γ cells without and with 70 nMof human IgE with binding ratios of 201-295 and to and human CD3expressed in Jurkat cells with binding ratios of 48-94. The exemplarybispecific antibodies of the invention showed minimal binding to HEK293without FcεR1 receptors with binding ratios of 2-11. Anti-FcεR1αantibodies showed binding to HEK293/hFcεR1α/hFcεR1β/hFcER1γ cellswithout and with 70 nM of human IgE with binding ratios of 136-396 andto HEK293 or Jurkat cells with binding ratios 1-8. Isotype controlantibodies showed no binding to any of the cells and secondary onlycontrols showed binding ratios of 1.

As shown in Table 14, exemplary anti-FcεR1α×CD3E bispecific antibodiesbsAb24919D, bsAb24920D, and bsAb24921D, showed binding to monkey(cynomolgus) FcεR1α expressed in HEK293/mfFcεR1α/hFcεR1β/hFcεR1γ cellswithout and with 70 nM of human IgE of 142-284. The exemplarybi-specific antibodies of the invention showed minimal binding to HEK293cells without FcεR1 receptors with binding ratios of 1-2. Exemplaryanti-FcεR1α antibodies of the invention showed binding toHEK293/mfFcεR1α/hFcεR1β/hFcεR1γ cells without and with 70 nM of humanIgE with binding ratios of 101-279 but not to HEK293. Isotype controlantibodies showed no binding to any of the cells and secondary onlycontrols showed binding ratios of 1.

Example 5: Activation of Human CD3 Signaling by Anti-FcεR1α×CD3Bispecific Antibodies

In order to assess the activation of human CD3 signaling byanti-FcεR1α×CD3E bi-specific antibodies in the presence of FcεR1αexpressing cells, a bioassay with Jurkat/NFAT-luc andHEK293/hFcεR1α/hFcεR1β/hFcεR1γ cells was performed. Stable cell lineswere developed. Jurkat cell line, a human T lymphocytic cell line, hasbeen utilized to demonstrate CD3 mediated T cell receptor signaling(Abraham and Weiss, Jurkat T cells and development of the T-cellreceptor signaling paradigm. Nat Rev Immunol. 2004 April; 4(4):301-8).Jurkat cells were engineered to stably express a luciferase reporterunder the transcription control of Nuclear factor of activated T-cells(NFAT) response element. The resulting cell line, referred to hereafteras Jurkat/NFAT-Luc was isolated and maintained in RPM11640 medium(Irvine Scientific, Cat. #9160) supplemented with 10% FBS, 1×Penicillin/Streptomycin/L-Glutamine and 1 g/mL Puromycin. Additionally,HEK293 cells were transfected to stably express human FcεR1α(amino acids1-257 of Uniprot #P12319-1), FcεR1β (amino acids 1-244 of Uniprot#001362-1) and FcεR1γ (amino acids 1-86 of Uniprot #P30273-1). Theresulting cell line, referred to hereafter asHEK293/hFcεR1α/hFcεR1β/hFcεR1γ was isolated and maintained in DMEMmedium (Irvine Science, Cat. #9033) supplemented with 10% FBS, 1×NEAA,1× Penicillin/Streptomycin/L-Glutamine, 1 g/mL Puromycin, 100 g/mL ofHygromycin B and 500 g/ml of G418 sulfate.

A bioassay was performed to measure the CD3 signaling by exemplaryanti-FcεR1α×CD3ε bi-specific antibodies of the invention. For thebioassay, HEK293 or HEK293/hFcεR1α/hFcεR1β/hFcεR1γ cells were plated at10,000 cells per well in a 96-well plate in assay buffer with or without10 nM of human IgE in assay buffer (10% FBS in RPM11640 (IrvineScientific, Cat #9160) with pen/strep/glut) for 30 minutes at 37° C. in5% CO₂. Following the incubation, Jurkat/NFAT-luc cells were plated at50,000 along with serially diluted exemplary anti-FcεR1α×CD3 bispecificantibodies of the invention, exemplary anti-FcεR1α of the invention orisotype control antibodies at concentrations ranging from 100 nM to 2 pMplus a sample containing buffer alone (no antibody). After 5.5 hours at37° C. in 5% CO₂, luciferase activity was measured with OneGlo™ reagent(Promega, #E6031) and Victor™ X multilabel plate reader (Perkin Elmer).The results were analyzed using nonlinear regression (4-parameterlogistics) with Prism™6 software (GraphPad) to obtain EC₅₀ values. Thefold activation was calculated with the average RLU (relative lightunits) at the highest concentration of antibody normalized by theaverage RLU without antibody. The results were summarized in Table 15.

TABLE 15 Activation of Human CD3 by Anti-FcεR1α × CD3 AntibodiesJurkat/NFAT-luc Jurkat/NFAT-luc HEK293/hFcεR1α /hFcεR1β/hFcεR1γ cellsHEK293 cells No IgE 10 nM IgE No IgE 10 nM IgE Fold Fold Antibody IDSpecificity EC₅₀ [M] EC₅₀ [M] EC₅₀ [M] Activation EC₅₀ [M] ActivationbsAb24919D FcεR1α × No No 3.4E−10 32 1.5E−09 32 hCD3 activationactivation bsAb24920D FcεR1α × No No 6.8E−10 23 4.9E−09 25 hCD3activation activation bsAb24921D FcεR1α × No No 2.3E−10 24 1.1E−09 27hCD3 activation activation mAb17110 FcεR1α No No No 1 No 1 activationactivation activation activation mAb17111 FcεR1α No No No 1 No 1activation activation activation activation mAb17112 FcεR1α No No No 1No 1 activation activation activation activation Human IgG4 StealthIrrelevant No No No 1 No 1 Control protein activation activationactivation activation Human lgG4 Control Irrelevant No No No 1 No 1protein activation activation activation activation

As shown in Table 15, exemplary anti-FcεR1α×CD3 bispecific antibodies ofthe invention, bsAb24919D, bsAb24920D, and bsAb24921D, showed activationof CD3 signaling in Jurkat/NFAT-luc cells with EC₅₀ values ranging from230 pM to 680 pM in the presence of HEK293/hFcεR1α/hFcεR1β/hFcεR1γ cellswithout human IgE and 1.1 nM to 4.9 nM with 10 nM of human IgE. Thehighest activation was achieved by bsAb24919D with fold activation of 32without and with 10 nM of IgE. The exemplary bispecific antibodies ofthe invention showed minimal activation in the presence of HEK293without FcεR1 receptors with fold activation ranging 1-3. Anti-FcεR1αand isotype control antibodies showed no activation with fold activationof 1 in any of the conditions tested.

Example 6: Effect of Anti-FcεR1α×Anti-CD3 Bispecific Antibodies in InVitro Killing Assays

To determine efficacy of exemplary anti-FcεR1α×anti-CD3 bispecificantibodies of the invention (bsAb24919D, bsAb24920D and bsAb24921 D) ininducing T cell-mediated killing of FcεR1α-expressing cells in vitro,two separate experiments were used. In one experiment, engineeredHEK293/hFcεR1α/hFcεR1β/hFcεR1γ cells were used as targets, while in thesecond experiment primary human basophils within a total peripheralblood mononuclear cell (PBMC) population were used as targets. In bothinstances similar protocols were used to activate T cells prior to thekilling assay: CD8+ T cells were first isolated from human leukopacks(NY Blood Center) using a RossetteSep™ Human CD8+ T cell enrichmentcocktail kit (STEMCELL Technologies, Cat. #15063) and placed in culturewith CD3/CD28-coated Dynabeads@ (Invitrogen, Cat. #11132D) to induceactivation of the T cells. On day 2-3 of culture, beads were removedusing magnetic separation and the T cells were placed in culture. In oneexample (for use with HEK293/hFcεR1α/hFcεR1β/hFcεR1γ target cells), Tcells were maintained in culture for 5 days, at which time IL-2 wasadded at 300 U/ml to promote viability and growth, and the T cells wereused 2 days after IL-2 addition. In the second example (for use withPBMC target cells), cells were maintained in culture for one day afterremoval of the beads and then used for the killing assay. In bothinstances, activated T cells were labeled with Carboxyfluoresceinsuccinimidyl ester (CFSE, Thermo Fischer Scientific, Cat. #C34554) priorto setting up the killing assay to enable exclusion of the cells duringanalysis of the results.

To determine efficacy of exemplary anti-FcεR1α×anti-CD3 bispecificantibodies of the invention in inducing T cell-mediated killingHEK293/hFcεR1α/hFcεR1β/hFcεR1γ target cells, a killing assay that usesdetection of two mediators of the apoptotic cascade as readout (cleavedcaspase 3 and cleaved PARP) was used. To set up the killing assay, theactivated T cells were mixed with the target cells at a ratio of 10target cells per T cell and then plated in a 96-well plate. Serialfive-fold antibody dilutions ranging in final concentration from 100 nMto 10.24 fM were added to the wells, and the cells were incubatedovernight at 37° C. to allow T cell-mediated killing to occur.Antibodies included exemplary anti-FcεR1α/CD3 bispecific antibodies ofthe invention and isotype control antibody. Following incubation, thecells were harvested and resuspended in pre-warmed BD cytofix (Cat.#554655) for 10 minutes at 37° C. Cells were then washed twice in MACSbuffer (Miltenyi, Cat. #130-091-221) and made permeable by resuspendingin ice-cold methanol and incubating at −20° C. for at least 30 minutesor overnight. Following permeabilization, MACS buffer was added to thecells for 10 minutes to allow cell rehydration, followed by 2 washeswith MACS buffer. Cells were then incubated with Fc-blocking antibody(Ebioscience, Cat. #14-9161-73), followed by staining with an antibodycocktail containing Alexa-647-conjugated anti-cleaved caspase 3 (CellSignaling Technology, Cat. #9602S) and a PE-conjugated anti-cleaved PARPantibodies (BD Biosciences, Cat. #552933). Cleavage of caspase 3 andPARP are obligatory steps in the activation of the apoptotic cascadethat is initiated after delivery of cytotoxic lytic granules from theCD8+ T cells to the targets. Thus, specific detection of these cleavedproteins serves as a readout of killing. After staining the cells werewashed, resuspended in MACS buffer and acquired using an LSRFortessainstrument (BD Biosciences). Killed cells were identified as CFSE-, andapoptotic cells within this population were identified as cleavedcaspase 3+ and cleaved PARP+. Data analysis was performed using GraphpadPrism software. The data points obtained were transformed using anX=Log(X) equation, and the transformed data were subjected to a linearregression analysis and fitted into a sigmoidal dose response curve.EC₈₀ (eighty percent (80%) of maximal effective concentration, whichincludes the concentration of an antibody which induces a eighty percent(80%) response between the baseline and maximum after a specifiedexposure time) and top responses were derived from this analysis.

To determine efficacy of exemplary anti-FcεR1α×anti-CD3 bispecificantibodies of the invention in inducing T cell-mediated killing ofprimary basophils within a peripheral blood mononuclear cell (PBMC)population, an assay based on quantitation of these cells relative tothe rest of the PBMC population was used. Fresh PBMCs were obtained fromdonor blood by Ficoll (GE Healthcare, Cat. #17-1440-03) purification andwere mixed with activated T cells and antibody dilutions in a similarformat as described above for the engineeredHEK293/hFcεR1α/hFcεR1β/hFcεR1γ target cells. After overnight incubation,cells were harvested, incubated with a Live/Dead cell marker (InvitrogenCat. #L34962), followed by incubation with an Fc-blocking antibody andstaining with an antibody mix containing APC-conjugated anti-HLA-DR (BDBiosciences, Cat. #559866) and BUV 395-conjugated anti-CD123 (BDBiosciences, Cat. #564195) antibodies. The cells were then washed twicewith MACS buffer and fixed in a solution containing BD Cytofix diluted1:4 in PBS for 15 minutes. Cells were resuspended in MACS buffer andacquired in a LSRFortessa instrument. Dead cells were excluded fromanalysis using the Live/Dead cell marker, as were exogenous activated Tcells that had previously been labeled with CFSE. Basophils within theremaining live PBMC population were identified as CD123+ HLA-DR-.

Data analysis was performed using Graphpad Prism software. The datapoints obtained were transformed using an X=Log(X) equation, and thetransformed data were subjected to a linear regression analysis andfitted into a sigmoidal dose response curve. EC₅₀s were derived fromthis analysis. Maximum percent basophil decrease was calculated usingthe following formula: 100−(100×percent basophils in sample with highestantibody dose)/(Average percent basophils in all isotype controlsamples).

Table 16 summarizes dose-dependent increases in cleaved caspase 3 andcleaved PARP double positive HEK293/hFcεR1α/hFcεR1β/hFcεR1γ target cellsin the presence of each exemplary bispecific antibodies of the invention(bsAb24919D, bsAb24920D and bsAb24921 D), with EC₈₀s of 3.456×10⁻¹¹ M,1.264×10⁻¹⁰ M, and 6.128×10⁻¹¹ M, respectively. Because this assay isbased on capturing the early stages of apoptosis, the assay is stoppedbefore the cells are fully killed, and the maximum percent of cellsstaining positive for the apoptotic markers was 56.04, 56.48 and 55.83for cells incubated with bsAb24919D, bsAb24920D and bsAb24921 D,respectively. Notably, no increases in the percentage of target cellspositive for both apoptotic markers were observed when T cells wereincubated together with the target cells in the absence of antibodyrelative to target cells incubated alone. In other words, induction ofapoptosis is not observed when T cells were incubated together with thetarget cells in the presence of isotype control antibody only.

TABLE 16 EC80 and Maximum Percent of Apoptotic Cells From Dose ResponseKilling Curves of HEK293/hFcεR1α/hFcεR1β/hFcεR1γ Target Cells afterIncubation with T Cells and FcεR1α × CD3 Bispecific AntibodiesbsAb24919D bsAb24920D bsAb24921D EC80 3.456e−11 1.264e−10 6.128e−11Maximum percent 56.04 56.48 55.83 apoptotic cells

Table 17 summarizes dose-dependent decreases in basophils within thetotal PBMC target population in the presence of exemplary bispecificantibodies of the invention (bsAb24919D, bsAb24920D and bsAb24921 D),with EC₅₀s of 3.748×10⁻⁹ M, 2.003×10⁻⁸ M, and 4.003×10⁻⁹ M,respectively. Basophils were decreased by 90.57%, 80.1% and 90.92% withthe highest dose of bsAb24919D, bsAb24920D and bsAb24921 D,respectively, relative to the average percent of basophils within thePBMCs in all isotype-treated samples.

TABLE 17 EC50 and Maximum Percent Basophil Decrease from Dose ResponseKilling Curves of Basophils within PBMC Target Cells after Incubationwith T Cells and Fcεr1α × CD3 Bispecific Antibodies bsAb24919DbsAb24920D bsAb24921D EC50 3.748e−9 2.003e−8 4.003e−9 Maximum percent90.57% 80.1% 90.92% basophil decrease

Example 7: In Vivo Efficacy of Anti-FcεR1α×CD3 Bispecific Antibodies

Effect of anti-FcεR1α×anti-CD3 bispecific antibodies in the passivecutaneous anaphylaxis (PCA) in vivo model and in splenic basophildepletion was studied.

To determine efficacy of anti-FcεR1α×CD3 bispecific antibodies of theinvention for blocking allergen induced mast cell degranulation, thepassive cutaneous anaphylaxis (PCA) in vivo model was used. The PCAmodel assesses type 1 hypersensitivity and measures local mast cellactivation-induced vascular permeability in ear tissue (Gilfillan, A. M.& Tkaczyk, C. Integrated signaling pathways for mast-cell activation.Nat. Rev. Immunol. 6, 218-230 (2006)). This model involves intradermalinjection of an allergen-specific sera from allergic patients into alocal area on the skin of mice that express the human high-affinity IgEreceptor, FcεR1α, followed by intravenous injection of an allergen alongwith a dye. The allergic reaction causes capillary dilatation andincreased vascular permeability at the site of sensitization, resultingin preferential accumulation of dye at this site. The dye can beextracted from the tissue and quantitated spectrophotometrically.

For the PCA assays, groups of mice humanized for FcεR1α and CD3 (n≥5 perexperiment) were first injected subcutaneously with either an isotypecontrol antibody or one of three exemplary FcεR1α×CD3 bispecificantibodies of the invention at a dose of 25 mg/kg. Five days afterantibody administration, mice were injected in the ear with serum from acat allergic individual (IgE titer 585, diluted 1:5 in PBS). Thefollowing day the mice were administered intravenously (100 μL permouse) a solution of 1 μg/mL Fel D1 (Indoor biotech LTN-FD1-1) dissolvedin 1×PBS containing 0.5% (w/v) Evan's blue dye (Sigma Aldrich, #E2129).One hour after antigen administration, mice were sacrificed, and theears and spleens were excised and collected.

The ears were placed in 1 mL formamide and subsequently incubated for 3days at 50° C. to extract the Evan's blue dye. The ear tissue was thenremoved from the formamide, blotted to remove excess liquid and weighed.Two-hundred microliter aliquots of each formamide extract weretransferred to 96 well plates in duplicate. Absorbance of the resultingsupernatants was measured at 620 nm. The optical density measured wasconverted to Evan's blue dye concentration using a standard curve and isrepresented as nanogram of Evan's blue dye per milligram ear tissue.Table 18 shows mean values±the standard deviation for each group.

To assess basophils frequency a flow cytometry-based assay was used.Single cell suspensions were prepared from the collected spleensfollowing red blood cell lysis (Sigma, Cat #R7757). The cells were thenstained with a live/dead cell marker, followed by antibody staining withthe antibody mixes containing BUV 395 conjugated anti-B220 (BD, Cat#563793), FITC conjugated anti-CD4 (BD, Cat #553031), FITC conjugatedanti-CD8 (BD, Cat #557667) and PECy7 conjugated CD49b (EBIOSCIENCE, Cat#25-5971-82). After staining, the cells were washed twice with MACSbuffer (Miltenyi Biotech Cat #130-091-221), fixed with BD Cytofix (Cat#554655) diluted 1:4 in PBS for 15 minutes, then resuspended in MACSbuffer and stored at 4 degrees. On the day of acquisition, the cellswere washed twice in BD Perm/wash buffer (Cat #554723) and stained forintracellular FcεR1α with the eFluor450 conjugated anti-FcεR1α(EBIOSCIENCE, Cat #48-5899-42). The cells were then acquired in anLSRFortessa instrument and analyzed using FlowJo software. Basophilswere identified as B220− CD4− CD8− CD49+ FcεR1α+. Percent reduction ofbasophils in individual antibody-administered mice was calculated withthe following formula: 100−(percent splenic basophils/mean percentsplenic basophils in the isotype group), where percent splenic basophilsare calculated relative to total live cells in the spleen. The resultsare shown in Table 19.

Evan's blue dye extravasation was observed in the ears of mice that werenot administered antibody or in those administered an isotype controlantibody, with a mean dye quantitation of 84.06 and 82.05 ng/mg,respectively (ng/mg refers to nanogram of Evan's blue dye per milligramof tissue). Table 18 demonstrates efficacy of the exemplaryanti-FcεR1α×CD3 bispecific antibodies of the invention (bsAb24919D andbsAb24921 D) in the PCA model as indicated by a significant reduction ofdye extravasation in the groups treated with these antibodies whencompared to isotype control. A non-statistically significant trendtowards reduced dye extravasation was observed in the group treated withbsAb24920D as compared to isotype control. As shown, bsAb24919D andbsAb24921 D block mast cell degranulation in the passive cutaneous invivo model against sensitization and subsequent challenge with Fel D1 ascompared to isotype control demonstrating a significant reduction in dyeextravasation of 74.64 ng/mg and 75.26 ng/mg respectively, while a moremodest reduction of 48.56 ng/mg was observed with bsAb24920D.Statistical significance was determined as follows: Normality of allgroups was first tested with the Shapiro-Wilk normality test. Becausethe data in all groups was normally distributed, a one-way ANOVAanalysis was applied, with a Brown-Forsythe test to determinedifferences in standard deviations across the groups. Significantlydifferent standard deviations were observed; thus, a non-parametricKruskal-Wallis test was run instead with Dunn's multiple comparison testto determine statistical significance among the groups.

Spleens from mice that were not administered antibody were found tocontain an average of 0.91% of basophils relative to total live cells,while those from mice administered isotype control antibody contained anaverage of 0.71% of basophils. Table 19 demonstrates efficacy ofexemplary FcεR1α×CD3 bispecific antibodies of the invention (bsAb24919D,bsAb24920D and bsAb24921 D) in depleting splenic basophils in the samemice from the PCA experiment described above. As shown, mice treatedwith any of the three antibodies showed a reduction in splenicbasophils. While this reduction was significant for all three antibodiesas compared to mice that were not administered antibody, the reductionwas only statistically significant for mice treated with bsAb24919D whencompared to the group administered an isotype control antibody. Micetreated with bsAb24919D, bsAb24920D and bsAb24921 D showed 96, 93 and 92percent reduction in basophils relative to the isotype group,respectively. Statistical significance was determined as follows: aShapiro-Wilk normality test was first run, and all data groups we foundto be normally distributed; thus, a one-way ANOVA analysis was applied,and a Brown-Forsythe test was used to test for differences in standarddeviations across the groups. Significantly different standarddeviations were observed in this test, so a non-parametricKruskal-Wallis test was run instead with Dunn's multiple comparison testto determine statistical significance among the groups.

TABLE 18 Effect of Anti-FcεR1α × CD3 Bispecific Antibodies in thePassive Cutaneous Anaphylaxis (PCA) in vivo Model ng Evans Blue/mg MeanDifference compared Treatment tissue ± SD to Isotype control bsAb24919D(n = 7) 7.41 ± 1.1  −74.64 (**) bsAb24920D (n = 7) 33.49 ± 16.54 −48.56(ns) bsAb24921D (n = 7) 6.79 ± 2.77 −75.26 (**) 25 mg/kg total antibodyconcentration used for all groups administered antibody *P ≤ .05, ***P ≤.001, ***P ≤ .0001 n = number of mice per group

TABLE 19 Effect of Anti-FcεR1α × CD3 Bispecific Antibodies in SplenicBasophil Depletion Mean Percent Decrease Splenic Basophils Relative toAverage (percent of Basophils in Isotype Treatment live cells) Controlgroup bsAb24919D (n = 7) 0.003 ± 0.0017 95.89 (**) bsAb24920D (n = 7)0.005 ± 0.0018 92.96 (ns) bsAb24921D (n = 7) 0.003 ± 0.0026 92.43 (ns)25 mg/kg total antibody concentration used for all groups administeredantibody *P ≤ .05, ***P ≤ .001, ***P ≤ .0001 n = number of mice pergroup

For both PCA and basophil depletion assays, ablation of cells expressingFcεR1α was achieved using exemplary anti-FcεR1α×CD3 bispecificantibodies of the invention at lower dosage. bsAb24919D and bsAb24921 Dshowed statistically significant inhibition of the PCA response andsignificant basophil loss in experiments repeated at lower doses of 1mg/kg, 5 mg/kg, and 10 mg/kg (data not shown). Efficacy is similar atdoses between 5 mg/kg and 25 mg/kg for both bsAb24919D and bsAb24921 Din both PCA and basophil depletion assays (data not shown).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A bispecific antigen-binding molecule comprising: a firstantigen-binding domain that specifically binds human CD3, and a secondantigen-binding domain that specifically binds human and/or cynomolgusFcεR1α; wherein the second antigen-binding domain comprises the heavyand light chain CDRs of an HCVR/LCVR amino acid sequence pair selectedfrom the group consisting of SEQ ID NOs: 2/26, 10/26, and 18/26. 2.-13.(canceled)
 14. The bispecific antigen-binding molecule of claim 1,wherein the second binding domain comprises a heavy chaincomplementarity determining region (HCDR)1 having the amino acidsequence of SEQ ID NO:4, an HCDR2 having the amino acid sequence of SEQID NO:6, an HCDR3 having the amino acid sequence of SEQ ID NO:8, a lightchain complementarity determining region (LCDR)1 having the amino acidsequence of SEQ ID NO:28, an LCDR2 having the amino acid sequence of SEQID NO:30, and an LCDR3 having the amino acid sequence of SEQ ID NO:32.15. The bispecific antigen-binding molecule of claim 14, wherein thesecond binding domain comprises an HCVR comprising the amino acidsequence of SEQ ID NO: 2 and an LCVR comprising the amino acid sequenceof SEQ ID NO:
 26. 16. The bispecific antigen-binding molecule of claim1, wherein the second binding domain comprises an HCDR1 having the aminoacid sequence of SEQ ID NO:12, an HCDR2 having the amino acid sequenceof SEQ ID NO:14, an HCDR3 having the amino acid sequence of SEQ IDNO:16, an LCDR1 having the amino acid sequence of SEQ ID NO:28, an LCDR2having the amino acid sequence of SEQ ID NO:30, and an LCDR3 having theamino acid sequence of SEQ ID NO:32.
 17. The bispecific antigen-bindingmolecule of claim 16, wherein the second binding domain comprises anHCVR comprising the amino acid sequence of SEQ ID NO: 10 and an LCVRcomprising the amino acid sequence of SEQ ID NO:
 26. 18. (canceled) 19.The bispecific antigen-binding molecule of claim 1, wherein the secondantigen-binding domain comprises an HCDR1 having the amino acid sequenceof SEQ ID NO:20, an HCDR2 having the amino acid sequence of SEQ IDNO:22, an HCDR3 having the amino acid sequence of SEQ ID NO:24, an LCDR1having the amino acid sequence of SEQ ID NO:28, an LCDR2 having theamino acid sequence of SEQ ID NO:30, and an LCDR3 having the amino acidsequence of SEQ ID NO:32.
 20. The bispecific antigen-binding molecule ofclaim 19, wherein the second antigen-binding domain comprises an HCVRcomprising the amino acid sequence of SEQ ID NO: 18 and an LCVRcomprising the amino acid sequence of SEQ ID NO:
 26. 21. (canceled) 22.The bispecific antigen-binding molecule of claim 1, wherein the firstantigen-binding domain that specifically binds human CD3 comprises heavychain complementarity determining regions (HCDR1, HCDR2 and HCDR3) froma heavy chain variable region (HCVR) comprising an amino acid sequenceof SEQ ID NO: 42, and light chain complementarity determining regions(LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR)comprising an amino acid sequence of SEQ ID NO:
 26. 23. The bispecificantigen-binding molecule of claim 1, wherein the first antigen-bindingdomain that specifically binds human CD3 comprises three heavy chaincomplementarity determining regions (HCDR1, HCDR2 and HCDR3) and threelight chain complementarity determining regions (LCDR1, LCDR2 andLCDR3), wherein HCDR1 comprises an amino acid sequence of SEQ ID NO: 44;HCDR2 comprises an amino acid sequence of SEQ ID NO: 46; HCDR3 comprisesan amino acid sequence of SEQ ID NO: 48; LCDR1 comprises an amino acidsequence of SEQ ID NO:28; LCDR2 comprises an amino acid sequence of SEQID NO:30; and LCDR3 comprises an amino acid sequence of SEQ ID NO:32.24. The bispecific antigen-binding molecule of claim 1, wherein thefirst antigen-binding domain that specifically binds human CD3 comprisesan HCVR/LCVR amino acid sequence pair of SEQ ID NO: 42/26.
 25. Thebispecific antigen-binding molecule of claim 1, wherein the firstantigen-binding domain that comprises HCDR1, HCDR2 and HCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 44, 46and 48 and LCDR1, LCDR2 and LCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 28, 30 and 32, wherein the firstantigen-binding domain binds human CD3; and wherein the secondantigen-binding domain comprises HCDR1, HCDR2 and HCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 4, 6,and 8, and LCDR1, LCDR2 and LCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 28, 30 and 32, wherein the secondantigen-binding domain binds human FcεR1α.
 26. The bispecificantigen-binding molecule of claim 1, wherein the first antigen-bindingdomain that comprises HCDR1, HCDR2 and HCDR3 domains, respectively,comprising the amino acid sequences of SEQ ID NOs: 44, 46 and 48 andLCDR1, LCDR2 and LCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 28, 30 and 32, wherein the firstantigen-binding domain binds human CD3; and wherein the secondantigen-binding domain comprises HCDR1, HCDR2 and HCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 12, 14and 16, and LCDR1, LCDR2 and LCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 28, 30 and 32, wherein the secondantigen-binding domain binds human FcεR1α.
 27. The bispecificantigen-binding molecule of claim 1, wherein the first antigen-bindingdomain that comprises HCDR1, HCDR2 and HCDR3 domains, respectively,comprising the amino acid sequences of SEQ ID NOs: 44, 46 and 48 andLCDR1, LCDR2 and LCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 28, 30 and 32, wherein the firstantigen-binding domain binds human CD3; and wherein the secondantigen-binding domain that comprises HCDR1, HCDR2 and HCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 20, 22and 24, and LCDR1, LCDR2 and LCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 28, 30 and 32, wherein the secondantigen-binding domain binds human FcεR1α. 28.-30. (canceled)
 31. Thebispecific antigen-binding molecule of claim 1 that is a bispecificantibody. 32.-35. (canceled)
 36. A nucleic acid molecule encoding thebispecific antigen-binding molecule of claim
 1. 37. A vector comprisingthe nucleic acid molecule of claim
 36. 38. A host cell comprising thevector of claim
 37. 39. A pharmaceutical composition comprising thebispecific antigen-binding molecule of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 40. A method of producing a bispecificantigen-binding molecule, the method comprising culturing the host cellof claim 38 under conditions permitting production of the bispecificantigen-binding molecule.
 41. A method for treating a disease ordisorder associated with FcεR1α expression and/or signaling in asubject, the method comprising administering to the subject a bispecificantigen-binding molecule comprising (i) a first antigen-binding domainthat specifically binds human CD3, and (i) a second antigen-bindingdomain that specifically binds human and/or cynomolgus FcεR1α: wherein:the first antigen-binding domain comprises the heavy and light chainCDRs of an HCVR/LCVR amino acid sequence pair of SEQ ID NO: 42/26; andthe second antigen-binding domain comprises the heavy and light chainCDRs of an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 2/26, 10/26, and 18/26.
 42. The method ofclaim 41, wherein the disease or disorder associated with FcεR1αexpression and/or signaling is an allergy, a mast cell activationdisorder, or mastocytosis.
 43. The method of claim 42, wherein theallergy is selected from the group consisting of allergic asthma, hayfever, atopic dermatitis, chronic urticaria, food allergy, and pollenallergy.
 44. The method of claim 42, wherein the allergy is ananaphylactic allergy.
 45. The method of claim 41, further comprisingadministering to the subject a second therapeutic agent.
 46. The methodof claim 45, wherein the second therapeutic agent is selected from thegroup consisting of an IgE antagonist, an anti-histamine, ananti-inflammatory agent, a corticosteroid, a leukotriene antagonist, amast cell inhibitor, a bronchial dilator, a decongestant, epinephrine,an IL-4 inhibitor, an IL-4 receptor inhibitor, an IL-33 antagonist, anIL-25 antagonist, a plasma cell ablating agent, and a TSLP antagonist.47.-49. (canceled)
 50. The method of claim 41, wherein the firstantigen-binding domain comprises a heavy chain complementaritydetermining region (HCDR)1 having the amino acid sequence of SEQ IDNO:44, an HCDR2 having the amino acid sequence of SEQ ID NO:46, an HCDR3having the amino acid sequence of SEQ ID NO:48, a light chaincomplementarity determining region (LCDR)1 having the amino acidsequence of SEQ ID NO:28, an LCDR2 having the amino acid sequence of SEQID NO:30, and an LCDR3 having the amino acid sequence of SEQ ID NO:32.51. The method of claim 50, wherein the first antigen-binding domaincomprises an HCVR comprising the amino acid sequence of SEQ ID NO: 42and an LCVR comprising the amino acid sequence of SEQ ID NO:
 26. 52. Themethod of claim 41, wherein the second antigen-binding domain comprisesan HCDR1 having the amino acid sequence of SEQ ID NO:4, an HCDR2 havingthe amino acid sequence of SEQ ID NO:6, an HCDR3 having the amino acidsequence of SEQ ID NO:8, an LCDR1 having the amino acid sequence of SEQID NO:28, an LCDR2 having the amino acid sequence of SEQ ID NO:30, andan LCDR3 having the amino acid sequence of SEQ ID NO:32.
 53. The methodof claim 52, wherein the second antigen-binding domain comprises an HCVRcomprising the amino acid sequence of SEQ ID NO: 2 and an LCVRcomprising the amino acid sequence of SEQ ID NO:
 26. 54. The method ofclaim 41, wherein the second antigen-binding domain comprises an HCDR1having the amino acid sequence of SEQ ID NO:12, an HCDR2 having theamino acid sequence of SEQ ID NO:14, an HCDR3 having the amino acidsequence of SEQ ID NO:16, an LCDR1 having the amino acid sequence of SEQID NO:28, an LCDR2 having the amino acid sequence of SEQ ID NO:30, andan LCDR3 having the amino acid sequence of SEQ ID NO:32.
 55. The methodof claim 54, wherein the second antigen-binding domain comprises an HCVRcomprising the amino acid sequence of SEQ ID NO: 10 and an LCVRcomprising the amino acid sequence of SEQ ID NO:
 26. 56. The method ofclaim 41, wherein the second antigen-binding domain comprises an HCDR1having the amino acid sequence of SEQ ID NO:20, an HCDR2 having theamino acid sequence of SEQ ID NO:22, an HCDR3 having the amino acidsequence of SEQ ID NO:24, an LCDR1 having the amino acid sequence of SEQID NO:28, an LCDR2 having the amino acid sequence of SEQ ID NO:30, andan LCDR3 having the amino acid sequence of SEQ ID NO:32.
 57. The methodof claim 56, wherein the second antigen-binding domain comprises an HCVRcomprising the amino acid sequence of SEQ ID NO: 18 and an LCVRcomprising the amino acid sequence of SEQ ID NO:
 26. 58. The method ofclaim 41, wherein the bispecific antigen-binding molecule is abispecific antibody that comprises a first heavy chain comprising theamino acid sequence of SEQ ID NO: 56, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO: 50, and a light chain comprisingan amino acid sequence of SEQ ID NO:
 40. 59. The method of claim 41,wherein the bispecific antigen-binding molecule is a bispecific antibodythat comprises a first heavy chain comprising the amino acid sequence ofSEQ ID NO: 56, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 52, and a light chain comprising an amino acid sequence ofSEQ ID NO:
 40. 60. The method of claim 41, wherein the bispecificantigen-binding molecule is a bispecific antibody that comprises a firstheavy chain comprising the amino acid sequence of SEQ ID NO: 56, asecond heavy chain comprising the amino acid sequence of SEQ ID NO: 54,and a light chain comprising an amino acid sequence of SEQ ID NO: 40.